Method of targeting senescent cells

ABSTRACT

A method of targeting a pharmaceutical agent to a senescent cell is disclosed. The method comprises administering the pharmaceutical agent to the subject, wherein said pharmaceutical agent is attached to an affinity moiety, said affinity moiety being capable of binding specifically to a polypeptide selected from the group consisting of HSP90B1, DNAJB4, PI4K2A, DBN1, PRKCSH, SPTBN1, NPM1, ITGA3 and a polypeptide set forth in Table 1. The targeting may be for therapeutics or diagnostics.

CROSS-REFERENCE

This application is a Continuation application of U.S. application Ser.No. 15/573,550 filed Nov. 13, 2017, which filed as a National Phase ofPCT Patent Application No. PCT/IL2016/050535 having International filingdate of May 19, 2016 and which claims the benefit of priority under 35USC § 119(e) of U.S. Provisional Patent Application No. 62/164,025 filedon May 20, 2015. The contents of the above applications are allincorporated by reference as if fully set forth herein in theirentirety.

SEQUENCE LISTING STATEMENT

The ASCII file, entitled P-587377-US1-SQL-09JAN19.txt, created on Jan.19, 2019, comprising 203,414 bytes, submitted concurrently with thefiling of this application is incorporated herein by reference.

FIELD AND BACKGROUND OF THE INVENTION

The present invention, in some embodiments thereof, relates to methodsof targeting senescent cells for treating and diagnosing diseases.

Cellular senescence, a stable form of cell cycle arrest, is a mechanismlimiting the proliferative potential of cells. Senescence can betriggered in many cell types in response to diverse forms of cellularstress. It is a potent barrier to tumorigenesis and contributes to thecytotoxicity of certain anti-cancer agents. While senescence limitstumorigenesis and tissue damage in a cell autonomous manner, senescentcells induce inflammation, tissue ageing, tissue destruction and promotetumorigenesis and metastasis in a cell non-autonomous manner in thesites of their presence. Therefore, their elimination might lead totumor prevention and inhibition of tissue ageing. Indeed, elimination ofsenescent cells was shown to slow down tissue ageing in an animal model(Baker et al., 2011).

Organisms may have developed elaborate mechanisms to eliminate senescentcells in order to avoid their deleterious effects on themicroenvironment. However, their fate in tissue is not wellcharacterized. On one hand, benign melanocytic nevi (moles) are highlyenriched for senescent cells yet can exist in skin throughout alifetime, implying that senescent cells can be stably incorporated intotissues. On the other hand, it has been previously shown that componentsof the innate immune system specifically recognize and eliminatesenescent cells in vitro and target senescent cells in vivo leading totumor regression and reversion of liver fibrosis (Krizhanovsky et al.,2008b; Sagiv et al., 2012; Xue et al., 2007). Therefore, senescent cellscan turn over in vivo and the immune system contributes to thisturnover. The effort that the immune system invests in recognition andelimination of senescent cells suggests, although not directly, thatsenescent cells are deleterious for the organism and their eliminationis beneficial.

Background art includes WO2014/174511, WO2013/152038, WO2014/089124, andGarnacho et al., Journal of Pharmacology and Experimental Therapeutics,March 2012 vol. 340 no. 3 638-647.

SUMMARY OF THE INVENTION

According to an aspect of some embodiments of the present inventionthere is provided a method of targeting a pharmaceutical agent to asenescent cell in a subject comprising administering the pharmaceuticalagent to the subject, wherein the pharmaceutical agent is attached to anaffinity moiety, the affinity moiety being capable of bindingspecifically to a polypeptide selected from the group consisting ofHSP90B1, DNAJB4, PI4K2A, DBN1, PRKCSH, SPTBN1, NPM1, ITGA3 and apolypeptide set forth in Table 1, thereby targeting the pharmaceuticalagent to the senescent cell.

According to an aspect of some embodiments of the present inventionthere is provided a method of treating a disease associated with cellsenescence in a subject in need thereof comprising administering to theagent a therapeutically effective amount of a cytotoxic agent attachedto an affinity moiety, the affinity moiety being capable of bindingspecifically to a polypeptide selected from the group consisting ofHSP90B1, DNAJB4, PI4K2A, DBN1, PRKCSH, SPTBN1, NPM1, ITGA3 and apolypeptide set forth in Table 1, thereby treating the disease.

According to an aspect of some embodiments of the present inventionthere is provided a particle having a senescent cell affinity moietyattached to an outer surface thereof, the senescent cell affinity moietycapable of specifically binding a polypeptide selected from the groupconsisting of HSP90B1, DNAJB4, PI4K2A, DBN1, PRKCSH, SPTBN1, NPM1, ITGA3and a polypeptide set forth in Table 1.

According to an aspect of some embodiments of the present inventionthere is provided a composition of matter comprising an affinity moietyattached to a therapeutic agent, wherein the affinity moietyspecifically binds to a polypeptide selected from the group consistingof HSP90B1, DNAJB4, PI4K2A, DBN1, PRKCSH, SPTBN1, NPM1, ITGA3 and apolypeptide set forth in Table 1.

According to an aspect of some embodiments of the present inventionthere is provided a pharmaceutical composition comprising thecomposition of matter or particle described herein as the active agentand a pharmaceutically acceptable carrier.

According to an aspect of some embodiments of the present inventionthere is provided a method of diagnosing a disease associated with cellsenescence in a subject comprising analyzing the amount of at least onepolypeptide selected from the group consisting of HSP90B1, DNAJB4,PI4K2A, DBN1, PRKCSH, SPTBN1, NPM1, ITGA3 and a polypeptide set forth inTable 1 on the membrane of cells of the subject, wherein a level of theat least one polypeptide above a predetermined amount is indicative ofthe disease.

According to an aspect of some embodiments of the present inventionthere is provided a method of identifying senescent cells in a cellpopulation comprising analyzing the amount of at least one polypeptideselected from the group consisting of HSP90B1, DNAJB4, PI4K2A, DBN1,PRKCSH, SPTBN1, NPM1, ITGA3 and a polypeptide set forth in Table 1 onthe membrane of the cells of the cell population, wherein a level of theat least one polypeptide is above a predetermined amount is indicativeof senescent cells.

According to an aspect of some embodiments of the present inventionthere is provided a composition of matter comprising senescent cells,wherein a polypeptide of the cells is attached to an affinity moiety,the polypeptide being selected from the group consisting of HSP90B1,DNAJB4, PI4K2A, DBN1, PRKCSH, SPTBN1, NPM1, ITGA3 and a polypeptide setforth in Table 1.

According to an aspect of some embodiments of the present inventionthere is provided a method of eliciting or boosting an immune responseto a senescent cell in a subject comprising administering to the subjecta pharmaceutical composition comprising at least one polypeptide or apolynucleotide encoding same selected from the group consisting ofHSP90B1, DBN1, PRKCSH, SPTBN1, NPM1 and a polypeptide set forth in Table1, wherein the composition does not comprise senescent cells ormembranes thereof, thereby eliciting or boosting the immune response tothe senescent cell.

According to an aspect of some embodiments of the present inventionthere is provided a vaccine comprising at least one polypeptide or apolynucleotide encoding same as an active agent, the polypeptide beingselected from the group consisting of HSP90B1, DBN1, PRKCSH, SPTBN1,NPM1, an adjuvant, wherein the vaccine does not comprise senescent cellsor membranes thereof and an immunologically acceptable carrier.

According to an aspect of some embodiments of the present inventionthere is provided a vaccine comprising cells expressing a heterogeneouspolypeptide, as an active agent and an immunologically acceptablecarrier, the polypeptide being selected from the group consisting ofHSP90B1, DBN1, PRKCSH, SPTBN1, NPM1.

According to some embodiments of the invention, the pharmaceutical agentis a therapeutic agent.

According to some embodiments of the invention, the therapeutic agent isa cytotoxic agent.

According to some embodiments of the invention, the pharmaceutical agentis a diagnostic agent.

According to some embodiments of the invention, the cytotoxic agent isdirectly attached to the affinity moiety.

According to some embodiments of the invention, the cytotoxic agent isindirectly attached to the affinity moiety.

According to some embodiments of the invention, the cytotoxic agent iscomprised in a particle.

According to some embodiments of the invention, the affinity moiety isattached to the outer surface of the particle.

According to some embodiments of the invention, the cytotoxic agentcomprises a polynucleotide agent.

According to some embodiments of the invention, the cytotoxic agentcomprises an RNA silencing agent.

According to some embodiments of the invention, the cytotoxic agentdown-regulates an activity and/or an amount of an apoptosis relatedpolypeptide.

According to some embodiments of the invention, the apoptosis relatedpolypeptide is selected from the group consisting of Bcl-xL, Bcl-w andp21.

According to some embodiments of the invention, the cytotoxic agent isselected from the group consisting of ABT-737, ABT-263, Gossypol,AT-101, TW-37 and Obatoclax.

According to some embodiments of the invention, the affinity moiety isselected from the group consisting or an antibody, an aptamer and apeptide.

According to some embodiments of the invention, the polypeptide isHSP90B1.

According to some embodiments of the invention, the disease is afibrotic disease or an inflammatory disease.

According to some embodiments of the invention, the inflammatory diseaseis cancer.

According to some embodiments of the invention, the method furthercomprises administering to the subject at least one agent selected fromthe group consisting of a sebum-regulating agent, an antibacterialand/or antifungal agent, a keratolytic agent and/or keratoregulatingagent, an astringent, an anti-inflammatory and/or anti-irritant, anantioxidant and/or free-radical scavenger, a cicatrizing agent, ananti-aging agent and a moisturizing agent.

According to some embodiments of the invention, the at least one agentis an anti-aging agent.

According to some embodiments of the invention, the affinity moiety isan antibody or an aptamer.

According to some embodiments of the invention, the particle is attachedto or encapsulating a therapeutic agent or a diagnostic agent.

According to some embodiments of the invention, the therapeutic agentcomprises a cytotoxic moiety.

According to some embodiments of the invention, the cytotoxic moietycomprises a polynucleotide agent.

According to some embodiments of the invention, the polynucleotide agentcomprises an RNA silencing agent.

According to some embodiments of the invention, the cytotoxic agentdown-regulates an activity and/or an amount of an apoptosis relatedpolypeptide.

According to some embodiments of the invention, the apoptosis relatedpolypeptide is selected from the group consisting of Bcl-xL, Bcl-w andp21.

According to some embodiments of the invention, the pharmaceuticalcomposition is formulated for topical administration.

According to some embodiments of the invention, the pharmaceuticalcomposition further comprises at least one agent selected from the groupconsisting of a sebum-regulating agent, an antibacterial and/orantifungal agent, a keratolytic agent and/or keratoregulating agent, anastringent, an anti-inflammatory and/or anti-irritant, an antioxidantand/or free-radical scavenger, a cicatrizing agent, an anti-aging agentand a moisturizing agent.

According to some embodiments of the invention, the at least one agentis an anti-aging agent.

According to some embodiments of the invention, the senescent cells arelysed cells.

According to some embodiments of the invention, the senescent cells arenon-lysed cells.

According to some embodiments of the invention, the disease is afibrotic disease or an inflammatory disease.

According to some embodiments of the invention, the inflammatory diseaseis cancer.

According to some embodiments of the invention, the identifying iseffected in vivo.

According to some embodiments of the invention, the identifying iseffected ex vivo.

According to some embodiments of the invention, the identifying iseffected in vitro.

According to some embodiments of the invention, the at least onepolypeptide is HSP90B1.

According to some embodiments of the invention, the analyzing iseffected using an antibody that selectively binds the at least onepolypeptide.

According to some embodiments of the invention, the antibody is attachedto a detectable moiety.

According to some embodiments of the invention, the vaccine furthercomprises an adjuvant.

According to some embodiments of the invention, the polypeptide isexpressed on a cell surface.

Unless otherwise defined, all technical and/or scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which the invention pertains. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of embodiments of the invention, exemplarymethods and/or materials are described below. In case of conflict, thepatent specification, including definitions, will control. In addition,the materials, methods, and examples are illustrative only and are notintended to be necessarily limiting.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

Some embodiments of the invention are herein described, by way ofexample only, with reference to the accompanying drawings and images.With specific reference now to the drawings in detail, it is stressedthat the particulars shown are by way of example and for purposes ofillustrative discussion of embodiments of the invention. In this regard,the description taken with the drawings makes apparent to those skilledin the art how embodiments of the invention may be practiced.

In the drawings:

FIGS. 1A-1E. Isolation and identification of senescence-specificmembrane proteins. (1A) Images of senescent IMR90 cells 9 days afterEtoposide (DIS), and senescent IMR90 cells 9 days after infection withH-Ras (OIS). Scale bar—100 μm. (1B) Schematic description of isolationand identification of membrane proteins. (1C-1E) Integrative analysis ofsenescence restricted cell surface proteins. (1C) Distribution ofidentified proteins in the two experiments (each experiment in at least3 repeats). The upper part shows distribution of proteins exclusivelydetected in each senescent cell type—DIS and OIS. The lower part showsprimary sub-cellular location of cell-surface proteome of senescentcells combined from both experiments. (1D) Immune-related canonicalpathways attributed to the identified proteins. (1E) Candidate upstreamregulators potentially responsible for the changes observed inidentified proteins as revealed by analysis using Ingenuity.

FIGS. 2A-2I. Grp94 is upregulated on the surface of senescent cells.(2A) Intersection of senescence specific cell-surface proteins from DISand OIS experiments, and the list of the nine shared proteins. (2B) Mainfeatures obtained from MS analysis regarding ICAM1, ITGA3, and Grp94.(2C) Location of unique peptides detected in MS analysis on the sequenceof Grp94 protein (in blue). (2D) Western blot analysis of Grp94, GAPDHand HLA-A,B,C from Qiagen surface-proteins isolation kit comparing tototal cell-lysate. (2E) Flow cytometry histogram for the frequency ofcell-surface Grp94 on live IMR90 cells. (2F) Quantitative analysis ofcell-surface Grp94 on live IMR90 cells. (2G) Flow cytometry histogramfor the frequency of cell-surface ICAM1 on live IMR90 cells. (2H)Quantitative analysis of cell-surface ICAM1 on live IMR90 cells. (2I)Immunofluorescent staining of live IMR90 cells for Grp94 (Alexa-647)against cell-surface marker HLA-A,B,C (Alexa-488). Scale bar—100 μm.

FIGS. 3A-3F. Grp94 translocates to cell-surface of senescent cells. (3A)Flow cytometry histogram for the frequency cell-surface Grp94 of liveIMR90 cells 0, 3, 7, 14, or 21 days after etoposide treatment. (3B)Quantitative analysis of cell-surface Grp94 on live IMR90 cells 0, 3, 7,14, or 21 days after etoposide treatment. (3C) Scheme describing themechanism of GPM1-mediated blockage of Grp94 translocation tocell-surface. (3D) PrestoBlue viability assay for IMR90 growing orsenescent Etoposide-treated cells after 24 hrs treatment with 1, 10, or100 μM GPM1. GPM1 does not affect cell viability. (3E) Flow cytometryhistogram for the frequency of cell-surface Grp94 of live senescentetoposide-treated IMR90 cells after 24 hrs treatment with 10 or 100 μMGPM1. (3F) Quantitative analysis of cell-surface Grp94 on live senescentetoposide-treated LMR90 cells after 24 hrs treatment with 10 or 100 μMGPM1.

FIGS. 4A-4E. Extracellular Grp94 on senescent cells mediate cytotoxicityof innate components in-vitro. (4A) Western blot analysis forGrp94/β-Tubulin in Etoposide-treated IMR90 cells 4 days aftertransfection with siControl or siHSP90B1 (HSP90B1 is a gene coding forGrp94). (4B) PrestoBlue viability assay for Etoposide-treated IMR90 4days after Transfection with siControl or siHSP90B1. (4C) Cytotoxicityof NK92 cells in cultures of IMR90 cells treated with siControl orsiHSP90B1. NK92 were added for 1 hour at a ratio of 1:3 (4D) CrystalViolet staining for IMR90 cells after co-culturing with MM6 monocytes inthe presence of GPM1. (4E) Quantitative analysis of cell-surface Grp94of Mouse embryonic fibroblasts (MEFs) 14 days after Etoposide treatmentcompared to growing MEFs.

FIGS. 5A-5D illustrate that GPM1 administration prevents immunesurveillance of senescent hepatic stellate cells in vivo. Mice treatedwith 12 intraperitoneal (i.p.) injections of CCl₄ (1 ml/kg, twice aweek) to induce liver fibrosis were subsequently subjected to 12 dailyi.p. injections of Vehicle or GPM1 (30 mg/kg/day), n=5. (5A) IF stainingon frozen sections of fibrotic livers treated with Vehicle or GPM1, forGrp94 (orange) and cell surface marker β-Catenin (green). Scale bar—20μm. (5B) SA-β-Gal staining (upper panel), H&E staining (middle panel),and Sirius Red staining (lower panel) of frozen sections from controlliver, fibrotic liver, and fibrotic livers after 12 days of Vehicle orGPM1. Scale bar of SA-β-Gal images—100 μm. Scale bar of Sirius Redimages—200 μm (5C) Quantification of SA-β-Gal positive cells in thelivers. Values are means+standard error of the mean (SEM). (5D)Quantification of fibrosis based on Sirius Red staining. Values aremeans+standard error of the mean (SEM). Fibrotic area in GPM1 treatedmice was compared to the one of Vehicle treated mice using Student's ttest (***p<0.01).

DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION

The present invention, in some embodiments thereof, relates to methodsof targeting senescent cells for treating and diagnosing diseases.

Before explaining at least one embodiment of the invention in detail, itis to be understood that the invention is not necessarily limited in itsapplication to the details set forth in the following description orexemplified by the Examples. The invention is capable of otherembodiments or of being practiced or carried out in various ways.

Cellular senescence, a stable irreversible cell-cycle arrest, preventspropagation of damaged cells in the organism. Senescent cells can befound in fibrotic or inflammatory diseases of skin, liver, lung,pancreas, prostate, as well as in articular cartilage, atheroscleroticplaques and other age-related diseases. Moreover, senescent cells wereshown to accumulate in normal tissues, especially skin, with age andsuggested to contribute to tissue ageing.

The immune system recognizes and eliminates senescent cells tofacilitate their removal from tissues. In order to unravel the molecularmechanisms behind this process, the present inventors evaluated the cellsurface proteome specific to human senescent fibroblasts. Nine proteinswere identified that were expressed exclusively on the surface ofsenescent cells (FIG. 2A). One of these proteins is glucose regulatedprotein 94 (Grp94), an ER chaperone which translocates to cell surface,and acts there as a potent regulator of the immune response.Cell-surface Grp94 was shown to accumulate in senescent cells in atime-dependent manner (FIG. 3A). This accumulation was inhibited byGPM1, a small-molecule which specifically promote Grp94 dimerization andretention in the ER.

Whilst reducing the present invention to practice, the present inventorsvalidated the presence of the full-size Grp94 protein on the surface ofsenescent cells (FIG. 2D), and evaluated its functional role in theinteraction with immune cells. The present inventors showed thatdown-regulation of cell-surface Grp94 decreases NK-cell mediatedcytotoxicity toward senescent cells. In a similar manner, GPM1 hasdecreased susceptibility of senescent cells for elimination bymonocytes.

The present inventors propose that the identified surface proteinsspecific for senescent cells may provide a target not only for theimmune system, but also for delivering specific agents to these cellsfor their labeling or elimination. The elimination of senescent cellsmight be valuable strategy to prevent cancer, treat cancer and treatvariety of age-related diseases where senescent cells are present.

Thus, according to a first aspect of the present invention there isprovided a method of targeting an agent to a senescent cell in a subjectcomprising administering the agent to the subject, wherein the agent isattached to an affinity moiety, the affinity moiety being capable ofbinding specifically to a polypeptide selected from the group consistingof HSP90B1, DNAJB4, PI4K2A, DBN1, PRKCSH, SPTBN1, NPM1, ITGA3 and apolypeptide set forth in Table 1, thereby targeting the agent to thesenescent cell.

The term “senescent cells” refers to cells that exhibit cell cyclearrest, generally during the G1 transition of the cell cycle or in fewcases in G2, elicited by replicative exhaustion due to telomereattrition or in response to stresses such as DNA damage,chemotherapeutic drugs, or aberrant expression of oncogenes.

According to a particular embodiment, the senescent cells arecharacterized by at least one or more of the following characteristics:

-   -   1. activation of the p53/p21CIP1 and/or pRb/p16INK4A tumor        suppressor pathways;    -   2. cells whose proliferation is irreversibly arrested;    -   3. shortening of telomere size;    -   4. expression of senescent-associated beta-galactosidase        activity;    -   5. Specific chromatin modification;    -   6. Specific secretome;    -   7. increase in reactive oxygen species and altered overall        mitochondrial activity.

Irreversible cell cycle arrest may be assessed by FACS or BrdUincorporation assay. Shortening of telomere size may be characterized byevaluating the mean terminal restriction fragment (TRF) length forexample by Southern blot analysis. Other methods of ascertaining whethera cell is senescent are described in US Patent No. 20140056860, thecontents of which are incorporated herein by reference.

Agents which may be targeted to the senescent cells include but are notlimited to therapeutic agents and diagnostic agents.

Exemplary therapeutic agents include nucleic acid, polypeptides e.g.antibodies, anticancer agent (e.g., chemotherapy, radioisotopes,immunotherapy), antibiotic, enzyme, antioxidant, lipid intake inhibitor,hormone, anti-inflammatory, steroid, vasodilator, angiotensin convertingenzyme inhibitor, angiotensin receptor antagonist, inhibitor for smoothmuscle cell growth and migration, platelet aggregation inhibitor,anticoagulant, inhibitor for release of chemical mediator, promoter orinhibitor for endothelial cell growth, aldose reductase inhibitor,inhibitor for mesangium cell growth, lipoxygenase inhibitor,immunosuppressive, immunostimulant, antiviral agent, Maillard reactionsuppressor, amyloidosis inhibitor, nitric oxide synthetic inhibitor,AGEs (Advanced glycation end-products) inhibitor, radical scavenger,protein, peptide; glycosaminoglycan and derivatives thereof; andoligosaccharide, polysaccharide, and derivatives thereof.

According to a particular embodiment, the pharmaceutical agent is acytotoxic agent.

As used herein, the term “cytotoxic agent” refers to refers to asubstance that inhibits or prevents the function of cells and/or causesdestruction of cells. The term is intended to include radioactiveisotopes (e.g., ²¹¹At, ¹³¹I, ¹²⁵I, ³²P, ³⁵S and radioactive isotopes ofLu, including ¹⁷⁷Lu, ⁸⁶Y, ⁹⁰Y, ¹¹¹In, ¹⁷⁷Lu, ²²⁵Ac, ²¹²Bi, ²¹³Bi, ⁶⁶Ga,⁶⁷Ga, ⁶⁸Ga, ⁶⁴Cu, ⁶⁷Cu, ⁷¹As, ⁷²As, ⁷⁶As, ⁷⁷As, ⁶⁵Zn, ⁴⁸V, ²⁰³Pb, ²⁰⁹Pb,²¹²Pb, ¹⁶⁶Ho, ¹⁴⁹Pm, ¹⁵³Sm, ²⁰¹Tl, ¹⁸⁸Re, ¹⁸⁶Re and ⁹⁹mTc), anticanceragents as otherwise described herein, including chemotherapeutic(anticancer drugs e.g. methotrexate, adriamicin, vinca alkaloids(vincristine, vinblastine, etoposide), taxol, doxoruicin, cisplatin,5-fluorouridine, melphalan, ethidium bromide, mitomycin C, chlorambucil,daunorubicin and other intercalating agents, enzymes and fragmentsthereof such as nucleolytic enzymes, antibiotics, therapeutic RNAmolecules (e.g., siRNA, antisense oligonucleotides, microRNA, ribozymes,RNA decoys, aptamers), DNAzymes, antibodies, proteins andpolynucleotides encoding same, and toxins such as small molecule toxinsor enzymatically active toxins of bacterial, fungal, plant or animalorigin, such as pokeweed antiviral protein (PAP), ricin toxin A, abrin,gelonin, saporin, cholera toxin A, diphtheria toxin, Pseudomonasexotoxin, and alpha-sarcin, including fragments and/or variants thereof.

As mentioned, the present invention contemplates the use of RNAsilencing agents as pharmaceutical agents and more specifically ascytotoxic agents. The RNA silencing agents may be directed againstanti-apoptotic proteins including but not limited to Bcl-xL, Bcl-wand/or p21. Other targets for RNA silencing are described inWO2013152038 and WO2014089124, the contents of which are incorporatedherein by reference.

The term “Bcl-xL” refers to the human protein also known as B-celllymphoma-extra large, having a sequence as set forth in SEQ ID NO: 1 andhomologs and orthologs thereof. The cDNA sequence of human Bcl-xL is setforth in SEQ ID NO: 2.

The term “Bcl-w” refers to the human protein also known as Bcl-2-likeprotein 2, having a sequence as set forth in SEQ ID NO: 3 and homologsand orthologs thereof. The cDNA sequence of human Bcl-w is set forth inSEQ ID NO: 4.

The term “p21” also known as “cyclin-dependent kinase inhibitor 1”refers to the human protein having a sequence as set forth in SEQ ID NO:5 and homologs and orthologs thereof. The cDNA sequence of human p21 isset forth in SEQ ID NO: 6.

As used herein, the phrase “RNA silencing” refers to a group ofregulatory mechanisms [e.g. RNA interference (RNAi), transcriptionalgene silencing (TGS), post-transcriptional gene silencing (PTGS),quelling, co-suppression, and translational repression] mediated by RNAmolecules which result in the inhibition or “silencing” of theexpression of a corresponding protein-coding gene. RNA silencing hasbeen observed in many types of organisms, including plants, animals, andfungi.

As used herein, the term “RNA silencing agent” refers to an RNA which iscapable of inhibiting or “silencing” the expression of a target gene. Incertain embodiments, the RNA silencing agent is capable of preventingcomplete processing (e.g, the full translation and/or expression) of anmRNA molecule through a post-transcriptional silencing mechanism. RNAsilencing agents include noncoding RNA molecules, for example RNAduplexes comprising paired strands, as well as precursor RNAs from whichsuch small non-coding RNAs can be generated. Exemplary RNA silencingagents include dsRNAs such as siRNAs, miRNAs and shRNAs. In oneembodiment, the RNA silencing agent is capable of inducing RNAinterference. In another embodiment, the RNA silencing agent is capableof mediating translational repression.

RNA interference refers to the process of sequence-specificpost-transcriptional gene silencing in animals mediated by shortinterfering RNAs (siRNAs). The corresponding process in plants iscommonly referred to as post-transcriptional gene silencing or RNAsilencing and is also referred to as quelling in fungi. The process ofpost-transcriptional gene silencing is thought to be anevolutionarily-conserved cellular defense mechanism used to prevent theexpression of foreign genes and is commonly shared by diverse flora andphyla. Such protection from foreign gene expression may have evolved inresponse to the production of double-stranded RNAs (dsRNAs) derived fromviral infection or from the random integration of transposon elementsinto a host genome via a cellular response that specifically destroyshomologous single-stranded RNA or viral genomic RNA.

The presence of long dsRNAs in cells stimulates the activity of aribonuclease III enzyme referred to as dicer. Dicer is involved in theprocessing of the dsRNA into short pieces of dsRNA known as shortinterfering RNAs (siRNAs). Short interfering RNAs derived from diceractivity are typically about 21 to about 23 nucleotides in length andcomprise about 19 base pair duplexes. The RNAi response also features anendonuclease complex, commonly referred to as an RNA-induced silencingcomplex (RISC), which mediates cleavage of single-stranded RNA havingsequence complementary to the antisense strand of the siRNA duplex.Cleavage of the target RNA takes place in the middle of the regioncomplementary to the antisense strand of the siRNA duplex.

Accordingly, the present invention contemplates use of dsRNA todownregulate protein expression from mRNA.

According to one embodiment, the dsRNA is greater than 30 bp. The use oflong dsRNAs (i.e. dsRNA greater than 30 bp) has been very limited owingto the belief that these longer regions of double stranded RNA willresult in the induction of the interferon and PKR response. However, theuse of long dsRNAs can provide numerous advantages in that the cell canselect the optimal silencing sequence alleviating the need to testnumerous siRNAs; long dsRNAs will allow for silencing libraries to haveless complexity than would be necessary for siRNAs; and, perhaps mostimportantly, long dsRNA could prevent viral escape mutations when usedas therapeutics.

Various studies demonstrate that long dsRNAs can be used to silence geneexpression without inducing the stress response or causing significantoff-target effects—see for example [Strat et al., Nucleic AcidsResearch, 2006, Vol. 34. No. 13 3803-3810; Bhargava A et al. Brain Res.Protoc. 2004; 13:115-125; Diallo M., et al., Oligonucleotides. 2003;13:381-392; Paddison P. J., et al., Proc. Natl Acad. Sci. USA. 2002;99:1443-1448; Tran N., et al., FEBS Lett. 2004; 573:127-134].

In particular, the present invention also contemplates introduction oflong dsRNA (over 30 base transcripts) for gene silencing in cells wherethe interferon pathway is not activated (e.g. embryonic cells andoocytes) see for example Billy et al., PNAS 2001, Vol 98, pages14428-14433 and Diallo et al, Oligonucleotides, Oct. 1, 2003, 13(5):381-392. doi:10.1089/154545703322617069.

The present invention also contemplates introduction of long dsRNAspecifically designed not to induce the interferon and PKR pathways fordown-regulating gene expression. For example, Shinagwa and Ishii [Genes& Dev. 17 (11): 1340-1345, 2003] have developed a vector, named pDECAP,to express long double-strand RNA from an RNA polymerase II (Pol II)promoter. Because the transcripts from pDECAP lack both the 5′-capstructure and the 3′-poly(A) tail that facilitate ds-RNA export to thecytoplasm, long ds-RNA from pDECAP does not induce the interferonresponse.

Another method of evading the interferon and PKR pathways in mammaliansystems is by introduction of small inhibitory RNAs (siRNAs) either viatransfection or endogenous expression.

The term “siRNA” refers to small inhibitory RNA duplexes (generallybetween 18-30 basepairs) that induce the RNA interference (RNAi)pathway. Typically, siRNAs are chemically synthesized as 21mers with acentral 19 bp duplex region and symmetric 2-base 3′-overhangs on thetermini, although it has been recently described that chemicallysynthesized RNA duplexes of 25-30 base length can have as much as a100-fold increase in potency compared with 21mers at the same location.The observed increased potency obtained using longer RNAs in triggeringRNAi is theorized to result from providing Dicer with a substrate(27mer) instead of a product (21mer) and that this improves the rate orefficiency of entry of the siRNA duplex into RISC.

It has been found that position of the 3′-overhang influences potency ofa siRNA and asymmetric duplexes having a 3′-overhang on the antisensestrand are generally more potent than those with the 3′-overhang on thesense strand (Rose et al., 2005). This can be attributed to asymmetricalstrand loading into RISC, as the opposite efficacy patterns are observedwhen targeting the antisense transcript.

It will be appreciated that more than one siRNA agent may be used todown-regulate a target gene. Thus, for example, the present inventioncontemplates use of at least two siRNAs that target Bcl-xL, at leastthree siRNAs that target Bcl-xL, or even at least four siRNAs thattarget Bcl-xL, each targeting a different sequence in the Bcl-xL gene.Further, the present invention contemplates use of at least two siRNAsthat target Bcl-w, at least three siRNAs that target Bcl-w, or even atleast four siRNAs that target Bcl-w, each targeting a different sequencein the Bcl-w gene. Further, the present invention contemplates use of atleast two siRNAs that target p21, at least three siRNAs that target p21,or even at least four siRNAs that target p21, each targeting a differentsequence in the p21 gene.

The strands of a double-stranded interfering RNA (e.g., a siRNA) may beconnected to form a hairpin or stem-loop structure (e.g., a shRNA).Thus, as mentioned the RNA silencing agent of the present invention mayalso be a short hairpin RNA (shRNA).

The term “shRNA”, as used herein, refers to an RNA agent having astem-loop stricture, comprising a first and second region ofcomplementary sequence, the degree of complementarity and orientation ofthe regions being sufficient such that base pairing occurs between theregions, the first and second regions being joined by a loop region, theloop resulting from a lack of base pairing between nucleotides (ornucleotide analogs) within the loop region. The number of nucleotides inthe loop is a number between and including 3 to 23, or 5 to 15, or 7 to13, or 4 to 9, or 9 to 11. Some of the nucleotides in the loop can beinvolved in base-pair interactions with other nucleotides in the loop.Examples of oligonucleotide sequences that can be used to form the loopinclude 5′-UUCAAGAGA-3′ (SEQ ID NO: 7; Brummelkamp, T. R. et al. (2002)Science 296: 550) and 5′-UUUGUGUAG-3′ (SEQ ID NO: 8; Castanotto, D. etal. (2002) RNA 8:1454). It will be recognized by one of skill in the artthat the resulting single chain oligonucleotide forms a stem-loop orhairpin structure comprising a double-stranded region capable ofinteracting with the RNAi machinery.

According to another embodiment the RNA silencing agent may be a miRNA.miRNAs are small RNAs made from genes encoding primary transcripts ofvarious sizes. They have been identified in both animals and plants. Theprimary transcript (termed the “pri-miRNA”) is processed through variousnucleolytic steps to a shorter precursor miRNA, or “pre-miRNA.” Thepre-miRNA is present in a folded form so that the final (mature) miRNAis present in a duplex, the two strands being referred to as the miRNA(the strand that will eventually basepair with the target) The pre-miRNAis a substrate for a form of dicer that removes the miRNA duplex fromthe precursor, after which, similarly to siRNAs, the duplex can be takeninto the RISC complex. It has been demonstrated that miRNAs can betransgenically expressed and be effective through expression of aprecursor form, rather than the entire primary form (Parizotto et al.(2004) Genes & Development 18:2237-2242 and Guo et al. (2005) Plant Cell17:1376-1386).

Unlike, siRNAs, miRNAs bind to transcript sequences with only partialcomplementarity (Zeng et al., 2002, Molec. Cell 9:1327-1333) and represstranslation without affecting steady-state RNA levels (Lee et al., 1993,Cell 75:843-854; Wightman et al., 1993, Cell 75:855-862). Both miRNAsand siRNAs are processed by Dicer and associate with components of theRNA-induced silencing complex (Hutvagner et al., 2001, Science293:834-838; Grishok et al., 2001, Cell 106: 23-34; Ketting et al.,2001, Genes Dev. 15:2654-2659; Williams et al., 2002, Proc. Natl. Acad.Sci. USA 99:6889-6894; Hammond et al., 2001, Science 293:1146-1150;Mourlatos et al., 2002, Genes Dev. 16:720-728). A recent report(Hutvagner et al., 2002, Sciencexpress 297:2056-2060) hypothesizes thatgene regulation through the miRNA pathway versus the siRNA pathway isdetermined solely by the degree of complementarity to the targettranscript. It is speculated that siRNAs with only partial identity tothe mRNA target will function in translational repression, similar to amiRNA, rather than triggering RNA degradation.

A suitable siRNA capable of downregulating Bcl-xL can be the siRNA ofSEQ ID NO: 9, 10 or 11. A suitable siRNA capable of downregulating Bcl-wcan be the siRNA of SEQ ID NO: 12, 13 or 14. A suitable siRNA capable ofdownregulating p21 can be the siRNA of SEQ ID NO: 15, 16 or 17.

It will be appreciated that the RNA silencing agent of the presentinvention need not be limited to those molecules containing only RNA,but further encompasses chemically-modified nucleotides andnon-nucleotides.

Other cytotoxic agents contemplated e present invention arepolynucleotides that encode pro-apoptotic proteins.

Non-limiting examples of pro-apoptotic genes include caspases, Bik,Puma, Bim, Bax, Bak, Bid, Bad, Bmf, Noxa, and Hrk.

The term “caspase” refers to proteases that play essential roles inapoptosis (programmed cell death) and necrosis. At least 12 caspaseshave been identified in humans. There are two types of apoptoticcaspases: initiator (apical) caspases and effector (executioner)caspases. Initiator caspases (e.g., CASP2 (Genbank Accession:NM001224.4), CASP8 (Genbank Accession: NM001080124.1), CASP9 (GenbankAccession: NM001229.3), and CASP10 (Genbank Accession: NM001206524.1))cleave inactive pro-forms of effector caspases, thereby activating them.Effector caspases (e.g., CASP3 (Genbank Accession: NM004346.3), CASP6(Genbank Accession: NM001226.3), CASP7 (Genbank Accession: NM001227.3))in turn cleave other protein substrates within the cell, to trigger theapoptotic process. The initiation of this cascade reaction is regulatedby caspase inhibitors.

Polynucleotide agents (e.g. encoding pro-apoptotic polypeptides orencoding an RNA silencing agent targeted against anti-apoptoticpolypeptides) are typically administered as part of an expressionconstruct. In this case, the polynucleotide agent is ligated in anucleic acid construct under the control of a cis-acting regulatoryelement (e.g. promoter) capable of directing an expression of thecytotoxic agent in a constitutive or inducible manner. An exemplarypromoter which is active in senescent cells is the p16 promoter—see forexample US Patent No. 20150064137, the contents of which areincorporated herein by reference.

The nucleic acid agent may be delivered using an appropriate genedelivery vehicle/method (transfection, transduction, etc.). Optionallyan appropriate expression system is used. Examples of suitableconstructs include, but are not limited to, pcDNA3, pcDNA3.1 (+/−),pGL3, PzeoSV2 (+/−), pDisplay, pEF/myc/cyto, pCMV/myc/cyto each of whichis commercially available from Invitrogen Co.(www(dot)invitrogen(dot)com).

The expression construct may also be a virus. Examples of viralconstructs include but are not limited to adenoviral vectors, retroviralvectors, vaccinia viral vectors, adeno-associated viral vectors, polyomaviral vectors, alphaviral vectors, rhabdoviral vectors, lenti viralvectors and herpesviral vectors.

A viral construct such as a retroviral construct includes at least onetranscriptional promoter/enhancer or locus-defining element(s), or otherelements that control gene expression by other means such as alternatesplicing, nuclear RNA export, or post-transcriptional modification ofmessenger. Such vector constructs also include a packaging signal, longterminal repeats (LTRs) or portions thereof, and positive and negativestrand primer binding sites appropriate to the virus used, unless it isalready present in the viral construct. In addition, such a constructtypically includes a signal sequence for secretion of the peptide from ahost cell in which it is placed. Preferably, the signal sequence forthis purpose is a mammalian signal sequence or the signal sequence ofthe peptide variants of the present invention. Optionally, the constructmay also include a signal that directs polyadenylation, as well as oneor more restriction site and a translation termination sequence. By wayof example, such constructs will typically include a 5′ LTR, a tRNAbinding site, a packaging signal, an origin of second-strand DNAsynthesis, and a 3′ LTR or a portion thereof.

Preferably the viral dose for infection is at least 10³, 10⁴, 10⁵, 10⁶,10⁷, 10⁸, 10⁹, 10¹⁰, 10¹¹, 10¹², 10¹³, 10¹⁴, 10¹⁵ or higher pfu or viralparticles.

Double stranded RNA may be synthesized by adding two opposing promotersto the ends of the gene segments, wherein one promoter is placedimmediately 5′ to the gene and the opposing promoter is placedimmediately 3′ to the gene segment. The dsRNA may then be transcribedwith the appropriate polymerase.

Other cytotoxic agents known to down-regulate anti-apoptotic proteinsare also contemplated by the present inventors—these include for exampleABT-737, ABT-263, Gossypol, AT-101, TW-37 and Obatoclax.

According to another embodiment, the pharmaceutical agent is adiagnostic agent.

Exemplary diagnostic drugs include in vivo diagnostics such as an X raycontrast medium, a diagnostic agent for ultrasound, an isotope-labeledagent for diagnosis by nuclear medicine, and an agent for diagnosis bynuclear magnetic resonance.

As mentioned, the pharmaceutical agents of this aspect of the presentinvention are attached either directly or indirectly to an affinitymoiety.

The affinity moiety may comprise a chemical (non-peptide) molecule, anaptamer, a peptide or an antibody (e.g. antibody-derived epitope bindingdomain) which is capable of specifically binding to one of the followingproteins:

HSP90B1, also referred to herein as Grp94, Refseq no: NM_003299.2, (SEQID NO: 18), DNAJB4 Refseq no: NM_007034.3 (SEQ ID NO: 19), PI4K2A Refseqno: NM_018425.3 (SEQ ID NO: 20), DBN1 Refseq no: NM_004395.3 orNM_080881.2 (SEQ ID NOs: 21), PRKCSH NM_001001329.2 or NM_001289102.1(SEQ ID NO: 22), SPTBN1 Refseq no: NM_178313.2 (SEQ ID NO: 23), NPM1Refseq no: NM_001037738.2 (SEQ ID NO: 24), ITGA3 NM_002204.2 (SEQ ID NO:25) and any of the polypeptides listed in Table 1 herein below.

For any of the aspects of the present invention, the polypeptide may beselected from the group consisting of HSP90B1, also referred to hereinas Grp94, Refseq no: NM_003299.2, (SEQ ID NO: 18), DNAJB4 Refseq no:NM_007034.3 (SEQ ID NO: 19), PI4K2A Refseq no: NM_018425.3 (SEQ ID NO:20), DBN1 Refseq no: NM_004395.3 or NM_080881.2 (SEQ ID NOs: 21), PRKCSHNM_001001329.2 or NM_001289102.1 (SEQ ID NO: 22), SPTBN1 Refseq no:NM_178313.2 (SEQ ID NO: 23), NPM1 Refseq no: NM_001037738.2 (SEQ ID NO:24), ITGA3 NM_002204.2 (SEQ ID NO: 25).

For any of the aspects of the present invention, the polypeptide may beselected from the group consisting of HSP90B1, also referred to hereinas Grp94, Refseq no: NM_003299.2, (SEQ ID NO: 18), DBN1 Refseq no:NM_004395.3 or NM_080881.2 (SEQ ID NOs: 21), PRKCSH NM_001001329.2 orNM_001289102.1 (SEQ ID NO: 22), SPTBN1 Refseq no: NM_178313.2 (SEQ IDNO: 23) and NPM1 Refseq no: NM_001037738.2 (SEQ NO: 24).

For any of the aspects of the present invention, the polypeptide may beHSP90B1, also referred to herein as Grp94, Refseq no: NM_003299.2, (SEQID NO: 18).

In one embodiment binding or specifically binding means a bindingaffinity (KD) of 10⁻⁸ mol/l or less, preferably 10⁻⁹ M to 10⁻¹³ mol/l.

In one embodiment, the affinity moiety binds to one of the aboveproteins with at least 5 fold higher affinity, 10 fold higher affinityor even 20 fold higher affinity than to a non-related protein.

The term “antibody” as used in this invention includes intact moleculesas well as functional fragments thereof.

As used herein, the phrase “antibody fragment” refers to a functionalfragment of an antibody (such as Fab, F(ab′)2, Fv, scFv, dsFv, or singledomain molecules such as VH and VL) that is capable of binding to anepitope of an antigen.

Suitable antibody fragments for practicing some embodiments of theinvention include a complementarity-determining region (CDR) of animmunoglobulin light chain (referred to herein as “light chain”), acomplementarity-determining region of an immunoglobulin heavy chain(referred to herein as “heavy chain”), a variable region of a lightchain, a variable region of a heavy chain, a light chain, a heavy chain,an Fd fragment, and antibody fragments comprising essentially wholevariable regions of both light and heavy chains such as a Fv, a singlechain Fv (scFv), a disulfide-stabilized Fv (dsFv), an Fab, an Fab′, andan F(ab′)2.

Functional antibody fragments comprising whole or essentially wholevariable regions of both light and heavy chains are defined as follows:

(i) Fv, defined as a genetically engineered fragment consisting of thevariable region of the light chain (VL) and the variable region of theheavy chain (VH) expressed as two chains;

(ii) single chain Fv (“scFv”), a genetically engineered single chainmolecule including the variable region of the light chain and thevariable region of the heavy chain, linked by a suitable polypeptidelinker as a genetically fused single chain molecule.

(iii) disulfide-stabilized Fv (“dsFv”), a genetically engineeredantibody including the variable region of the light chain and thevariable region of the heavy chain, linked by a genetically engineereddisulfide bond.

(iv) Fab, a fragment of an antibody molecule containing a monovalentantigen-binding portion of an antibody molecule which can be obtained bytreating whole antibody with the enzyme papain to yield the intact lightchain and the Fd fragment of the heavy chain which consists of thevariable and CH1 domains thereof;

(v) Fab′, a fragment of an antibody molecule containing a monovalentantigen-binding portion of an antibody molecule which can be obtained bytreating whole antibody with the enzyme pepsin, followed by reduction(two Fab′ fragments are obtained per antibody molecule);

(vi) F(ab′)2, a fragment of an antibody molecule containing a monovalentantigen-binding portion of an antibody molecule which can be obtained bytreating whole antibody with the enzyme pepsin (i.e., a dimer of Fab′fragments held together by two disulfide bonds); and

(vii) Single domain antibodies are composed of a single VH or VL domainswhich exhibit sufficient affinity to the antigen.

Methods of producing polyclonal and monoclonal antibodies as well asfragments thereof are well known in the art (See for example, Harlow andLane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory,New York, 1988, incorporated herein by reference).

Antibody fragments according to some embodiments of the invention can beprepared by proteolytic hydrolysis of the antibody or by expression inE. coli or mammalian cells (e.g. Chinese hamster ovary cell culture orother protein expression systems) of DNA encoding the fragment. Antibodyfragments can be obtained by pepsin or papain digestion of wholeantibodies by conventional methods. For example, antibody fragments canbe produced by enzymatic cleavage of antibodies with pepsin to provide a5S fragment denoted F(ab′)2. This fragment can be further cleaved usinga thiol reducing agent, and optionally a blocking group for thesulfhydryl groups resulting from cleavage of disulfide linkages, toproduce 3.5S Fab′ monovalent fragments. Alternatively, an enzymaticcleavage using pepsin produces two monovalent Fab′ fragments and an Fcfragment directly. These methods are described, for example, byGoldenberg, U.S. Pat. Nos. 4,036,945 and 4,331,647, and referencescontained therein, which patents are hereby incorporated by reference intheir entirety. See also Porter, R. R. [Biochem. J. 73: 119-126 (1959)].Other methods of cleaving antibodies, such as separation of heavy chainsto form monovalent light-heavy chain fragments, further cleavage offragments, or other enzymatic, chemical, or genetic techniques may alsobe used, so long as the fragments bind to the antigen that is recognizedby the intact antibody.

Fv fragments comprise an association of VH and VL chains. Thisassociation may be noncovalent, as described in Inbar et al. [Proc.Nat'l Acad. Sci. USA 69:2659-62 (19720]. Alternatively, the variablechains can be linked by an intermolecular disulfide bond or cross-linkedby chemicals such as glutaraldehyde. Preferably, the Fv fragmentscomprise VH and VL chains connected by a peptide linker. Thesesingle-chain antigen binding proteins (sFv) are prepared by constructinga structural gene comprising DNA sequences encoding the VH and VLdomains connected by an oligonucleotide. The structural gene is insertedinto an expression vector, which is subsequently introduced into a hostcell such as E. coli. The recombinant host cells synthesize a singlepolypeptide chain with a linker peptide bridging the two V domains.Methods for producing sFvs are described, for example, by [Whitlow andFilpula, Methods 2: 97-105 (1991); Bird et al., Science 242:423-426(1988); Pack et al., Bio/Technology 11:1271-77 (1993); and U.S. Pat. No.4,946,778, which is hereby incorporated by reference in its entirety.

Another form of an antibody fragment is a peptide coding for a singlecomplementarity-determining region (CDR). CDR peptides (“minimalrecognition units”) can be obtained by constructing genes encoding theCDR of an antibody of interest. Such genes are prepared, for example, byusing the polymerase chain reaction to synthesize the variable regionfrom RNA of antibody-producing cells. See, for example, Larrick and Fry[Methods, 2: 106-10 (1991)].

Humanized forms of non-human (e.g., murine) antibodies are chimericmolecules of immunoglobulins, immunoglobulin chains or fragments thereof(such as Fv, Fab, Fab′, F(ab′).sub.2 or other antigen-bindingsubsequences of antibodies) which contain minimal sequence derived fromnon-human immunoglobulin. Humanized antibodies include human isimmunoglobulins (recipient antibody) in which residues form acomplementary determining region (CDR) of the recipient are replaced byresidues from a CDR of a non-human species (donor antibody) such asmouse, rat or rabbit having the desired specificity, affinity andcapacity. In some instances, Fv framework residues of the humanimmunoglobulin are replaced by corresponding non-human residues.Humanized antibodies may also comprise residues which are found neitherin the recipient antibody nor in the imported CDR or frameworksequences. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the CDR regions correspond to thoseof a non-human immunoglobulin and all or substantially all of the FRregions are those of a human immunoglobulin consensus sequence. Thehumanized antibody optimally also will comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin [Jones et al., Nature, 321:522-525 (1986); Riechmann etal., Nature, 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol.,2:593-596 (1992)].

Methods for humanizing non-human antibodies are well known in the art.Generally, a humanized antibody has one or more amino acid residuesintroduced into it from a source which is non-human. These non-humanamino acid residues are often referred to as import residues, which aretypically taken from an import variable domain. Humanization can beessentially performed following the method of Winter and co-workers[Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)], bysubstituting rodent CDRs or CDR sequences for the correspondingsequences of a human antibody. Accordingly, such humanized antibodiesare chimeric antibodies (U.S. Pat. No. 4,816,567), wherein substantiallyless than an intact human variable domain has been substituted by thecorresponding sequence from a non-human species. In practice, humanizedantibodies are typically human antibodies in which some CDR residues andpossibly some FR residues are substituted by residues from analogoussites in rodent antibodies.

Human antibodies can also be produced using various techniques known inthe art, including phage display libraries [Hoogenboom and Winter, J.Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581(1991)]. The techniques of Cole et al. and Boerner et al. are alsoavailable for the preparation of human monoclonal antibodies (Cole etal., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77(1985) and Boerner et al., J. Immunol., 147(1):86-95 (1991)]. Similarly,human antibodies can be made by introduction of human immunoglobulinloci into transgenic animals, e.g., mice in which the endogenousimmunoglobulin genes have been partially or completely inactivated. Uponchallenge, human antibody production is observed, which closelyresembles that seen in humans in all respects, including generearrangement, assembly, and antibody repertoire. This approach isdescribed, for example, in U.S. Pat. Nos. 5,545,807; 5,545,806;5,569,825; 5,625,126; 5,633,425; 5,661,016, and in the followingscientific publications: Marks et al., Bio/Technology 10: 779-783(1992); Lonberg et al., Nature 368: 856-859 (1994); Morrison, Nature 368812-13 (1994); Fishwild et al., Nature Biotechnology 14, 845-51 (1996);Neuberger, Nature Biotechnology 14: 826 (1996); and Lonberg and Huszar,Intern. Rev. Immunol. 13, 65-93 (1995).

Antibodies directed against any of the proteins HSP90B1, DNAJB4, PI4K2A,DBN1, PRKCSH, SPTBN1, NPM1, ITGA3 and those listed in Table 1 arecommercially available from Pierce (e.g. anti-Grp94; PA5-24824).

As mentioned, the affinity moiety of this aspect of the presentinvention may also be an aptamer.

As used herein, the term “aptamer” refers to a nucleic acid thatspecifically binds to a target, such as a protein, through interactionsother than Watson-Crick base pairing. In a particular embodiment, theaptamer specifically binds to one or more targets (e.g., a protein orprotein complex) to the general exclusion of other molecules in asample. The aptamer may be a nucleic acid such as an RNA, a DNA, amodified nucleic acid, or a mixture thereof. The aptamer may also be anucleic acid in a linear or circular form and may be single stranded ordouble stranded. The aptamer may comprise oligonucleotides that are atleast 5, at least 10, at least 15, at least 20, at least 25, at least30, at least 35, at least 40 or more nucleotides in length. Aptamers maycomprise sequences that are up to 40, up to 60, up to 80, up to 100, upto 150, up to 200 or more nucleotides in length. Aptamers may be fromabout 5 to about 150 nucleotides, from about 10 to about 100nucleotides, or from about 20 to about 75 nucleotides in length. Whileaptamers are discussed herein as nucleic acid molecules (e.g.,oligonucleotides) aptamers, aptamer equivalents may also be used inplace of the nucleic acid aptamers, such as peptide aptamers.

According to one embodiment, the pharmaceutical agent and the affinitymoiety (e.g. antibody or aptamer) are attached directly to one another.

Thus according to another aspect of the present invention there isprovided a composition of matter (e.g. complex) comprising an affinitymoiety attached to a therapeutic agent, wherein the affinity moietyspecifically binds to a polypeptide selected from the group consistingof HSP90B1, DNAJB4, PI4K2A, DBN1, PRKCSH, SPTBN1, NPM1, ITGA3 and any ofthe polypeptides listed in Table 1, herein below.

Affinity moieties have been described herein above.

The pharmaceutical agent of the invention may be attached or conjugatedto the affinity moiety of the invention in various ways, depending onthe context, application and purpose.

When both the pharmaceutical agent and the affinity moiety arepolypeptides, the conjugate may be produced by recombinant means. Forexample, the nucleic acid sequence encoding a toxin (e.g., PE38KDEL) ora fluorescent protein [e.g., green fluorescent protein (GFP), redfluorescent protein (RFP) or yellow fluorescent protein (YFP)] may beligated in-frame with the nucleic acid sequence encoding an antibody ofthe invention and be expressed in a host cell to produce a recombinantconjugated antibody. Alternatively, at least one of the affinity moietyor pharmaceutical agent may be chemically synthesized by, for example,the stepwise addition of one or more amino acid residues in definedorder such as solid phase peptide synthetic techniques.

A pharmaceutical agent may also be attached to the affinity moiety ofthe invention using standard chemical synthesis techniques widelypracticed in the art [see e.g., hypertexttransferprotocol://worldwideweb(dot) chemistry (dot) org/portal/Chemistry)], such as using any suitablechemical linkage, direct or indirect, as via a peptide bond (when thefunctional moiety is a polypeptide), or via covalent bonding to anintervening linker element, such as a linker peptide or other chemicalmoiety, such as an organic polymer. Chimeric peptides may be linked viabonding at the carboxy (C) or amino (N) termini of the peptides, or viabonding to internal chemical groups such as straight, branched or cyclicside chains, internal carbon or nitrogen atoms, and the like.

Exemplary methods for conjugating peptide pharmaceutical agent topolypeptide affinity moieties (e.g. antibodies) are described hereinbelow:

SPDP conjugation—A non-limiting example of a method of SPDP conjugationis described in Cumber et al. (1985, Methods of Enzymology 112:207-224). Briefly, a peptide, such as a detectable or therapeutic moiety(e.g., 1.7 mg/ml) is mixed with a 10-fold excess of SPDP (50 mM inethanol); the antibody is mixed with a 25-fold excess of SPDP in 20 mMsodium phosphate, 0.10 M NaCl pH 7.2 and each of the reactions isincubated for about 3 hours at room temperature. The reactions are thendialyzed against PBS. The peptide is reduced, e.g., with 50 mM DTT for 1hour at room temperature. The reduced peptide is desalted byequilibration on G-25 column (up to 5% sample/column volume) with 50 mMKH₂PO₄ pH 6.5. The reduced peptide is combined with the SPDP-antibody ina molar ratio of 1:10 antibody:peptide and incubated at 4° C. overnightto form a peptide-antibody conjugate.

Glutaraldehyde conjugation—A non-limiting example of a method ofglutaraldehyde conjugation is described in G. T. Hermanson (1996,“Antibody Modification and Conjugation, in Biconjugate Techniques,Academic Press, San Diego). Briefly, the antibody and the peptide (1.1mg/ml) are mixed at a 10-fold excess with 0.05% glutaraldehyde in 0.1 Mphosphate, 0.15 M NaCl pH 6.8, and allowed to react for 2 hours at roomtemperature. 0.01 M lysine can be added to block excess sites. After-thereaction, the excess glutaraldehyde is removed using a G-25 columnequilibrated with PBS (10% v/v sample/column volumes).

Carbodiimide conjugation—Conjugation of a peptide with an antibody canbe accomplished using a dehydrating agent such as a carbodiimide, e.g.,in the presence of 4-dimethyl aminopyridine. Carbodiimide conjugationcan be used to form a covalent bond between a carboxyl group of peptideand an hydroxyl group of an antibody (resulting in the formation of anester bond), or an amino group of an antibody (resulting in theformation of an amide bond) or a sulfhydryl group of an antibody(resulting in the formation of a thioester bond). Likewise, carbodiimidecoupling can be used to form analogous covalent bonds between a carbongroup of an antibody and an hydroxyl, amino or sulfhydryl group of thepeptide [see, J. March, Advanced Organic Chemistry: Reaction's,Mechanism, and Structure, pp. 349-50 & 372-74 (3d ed.), 1985]. Forexample, the peptide can be conjugated to an antibody via a covalentbond using a carbodiimide, such as dicyclohexylcarbodiimide [B. Neiseset al. (1978), Angew Chem., Int. Ed. Engl. 17:522; A. Hassner et al.(1978, Tetrahedron Lett. 4475); E. P. Boden et al. (1986, J. Org. Chem.50:2394) and L. J. Mathias (1979, Synthesis 561)].

When both the pharmaceutical agent and the affinity moiety areantibodies, the present invention contemplates generation of bispecificantibodies wherein each arm of the antibody recognizes a differentantigen.

It will be appreciated that the affinity moiety and the pharmaceuticalagent of this aspect of the present invention may be attachedindirectly—e.g. via a particle, wherein the pharmaceutical agent isinside the particle or on the outer surface thereof and the affinitymoiety is on the outer surface of the particle.

Thus, according to another aspect of the present invention there isprovided a particle having a senescent cell affinity moiety attached toan outer surface thereof, the senescent cell affinity moiety capable ofspecifically binding a polypeptide selected from the group consisting ofHSP90B1, DNAJB4, PI4K2A, DBN1, PRKCSH, SPTBN1, NPM1, ITGA3 and any ofthe polypeptides listed in Table 1, herein below.

Senescent cell affinity moieties have been described herein above.

As used herein, “particles” refers to nano—micro structures which arenot biological cells.

The particle may be a synthetic carrier, gel or other object or materialhaving an external surface which is capable of being loadable with(e.g., encapsulating) a pharmaceutical agent. The particle may be eitherpolymeric or non-polymeric preparations.

Exemplary particles that may be used according to this aspect of thepresent invention include, but are not limited to polymeric particles,microcapsules, liposomes, microspheres, microemulsions, nanoparticles,nanocapsules, nano-spheres, nano-liposomes, nano-emulsions andnanotubes.

According to a particular embodiment, the particles are nanoparticles.

As used herein, the term “nanoparticle” refers to a particle orparticles having an intermediate size between individual atoms andmacroscopic bulk solids. Generally, nanoparticle has a characteristicsize (e.g., diameter for generally spherical nanoparticles, or lengthfor generally elongated nanoparticles) in the sub-micrometer range,e.g., from about 1 nm to about 500 nm, or from about 1 nm to about 200nm, or of the order of 10 nm, e.g., from about 1 nm to about 100 nm. Thenanoparticles may be of any shape, including, without limitation,elongated particle shapes, such as nanowires, or irregular shapes, inaddition to more regular shapes, such as generally spherical, hexagonaland cubic nanoparticles. According to one embodiment, the nanoparticlesare generally spherical.

The particles of this aspect of the present invention may have a chargedsurface (i.e., positively charged or negatively charged) or a neutralsurface.

Agents which are used to fabricate the particles may be selectedaccording to the desired charge required on the outer surface of theparticles.

Thus, for example if a negatively charged surface is desired, theparticles may be fabricated from negatively charged lipids (i.e. anionicphospholipids) such as described herein below.

When a positively charged surface is desired, the particles may befabricated from positively charged lipids (i.e. cationic phospholipids),such as described herein below.

As mentioned, non charged particles are also contemplated by the presentinvention. Such particles may be fabricated from neutral lipids such asphosphatidylethanolamine or dioleoylphosphatidylethanolamine (DOPE).

It will be appreciated that combinations of different lipids may be usedto fabricate the particles of the present invention, including a mixtureof more than one cationic lipid, a mixture of more than one anioniclipid, a mixture of more than one neutral lipid, a mixture of at leastone cationic lipid and at least one anionic lipid, a mixture of at leastone cationic lipid and at least one neutral lipid, a mixture of at leastone anionic lipid and at least one neutral lipid and additionalcombinations of the above. In addition, polymer-lipid based formulationsmay be used.

There are numerous polymers which may be attached to lipids. Polymerstypically used as lipid modifiers include, without being limitedthereto: polyethylene glycol (PEG), polysialic acid, polylactic (alsotermed polylactide), polyglycolic acid (also termed polyglycolide),poly-(lactic-co-glycolic)poly-(vinyl-alcohol), polyvinylpyrrolidone,polyethyloxazoline, polyllydroxyetlyloxazolille,solyhydroxypryloxazoline, polyhydroxypropyl methacrylamide,polymethacrylamide, polydimethylacrylamide, polyvinylmethylether,polyhydroxyethyl acrylate, derivatized celluloses such ashydroxymethylcellulose or hydroxyethylcellulose.

The polymers may be employed as homopolymers or as block or randomcopolymers.

The particles may also include other components. Examples of such othercomponents includes, without being limited thereto, fatty alcohols,fatty acids, and/or cholesterol esters or any other pharmaceuticallyacceptable excipients which may affect the surface charge, the membranefluidity and assist in the incorporation of the biologically activelipid into the lipid assembly. Examples of sterols include cholesterol,cholesterol hemisuccinate, cholesterol sulfate, or any other derivativesof cholesterol. Preferred lipid assemblies according the inventioninclude either those which form a micelle (typically when the assemblyis absent from a lipid matrix) or those which form a liposome(typically, when a lipid matrix is present).

The particles of the present invention may be modified. Accordingmodified to enhance their circulatory half-life (e.g. by PEGylation) toreduce their clearance, to prolong their scavenging time-frame and toallow antibody binding. The PEG which is incorporated into the articlesmay be characterized by of any of various combinations of chemicalcomposition and/or molecular weight, depending on the application andpurpose.

Methods of coupling affinity moieties (e.g. antibodies) on particle'souter surface (e.g., liposomes) are known in the art.

As used herein “coupling” or “coupled on” refers to covalent ornon-covalent attachment of the affinity moiety to the particle.

Antibody conjugation methods which can be used in accordance with theteachings of the present invention can be divided to direct binding orindirect binding. Some methods are provided hereinbelow and aresummarized in Ansell, Supra. While specifically referring to liposomes,the procedures described hereinbelow may be applied to a variety ofparticles, while using modified protocols simply applied by the ordinaryartisan.

Direct conjugation methods are well known to those of skill in the art.See for example, G. Gregoriadis, (1984) “Liposome Technology” CRC Press,Boca Raton, Fla. and D. D. Lasic, “Liposomes: from physics toapplications” (1993) Elsevier, Amsterdam; N.Y. Particularly preferred isconjugation through a thioether linkage. This may be accomplished byreacting the antibody with a maleimide derivatized lipid such asmaleimide derivatized phosphatidylethanolamine (M-PE) ordipalmitoylethanolamine (M-DEP). This approach is described in detail byMartin et al. J. Biol. Chem., 257: 286-288 (1982) which is incorporatedherein by reference.

In another preferred embodiment, the antibody can be coupled to ahydrophilic polymer (e.g., a PEG). Means of attaching targetingmolecules to polymer linkers are well known to those of skill in the art(see, e.g., chapter 4 in Monoclonal Antibodies: Principles andApplications, Birch and Lennox, eds., John Wiley & Sons, Inc., New York(1995); and Blume et al. Biochem. Biophys. Acta. 1149: 180-184 (1993).In a particularly preferred embodiment, an antibody or a fragmentthereof (e.g., Fab′ fragment) is linked to a maleimide derivatized PEGthrough the —SII group of the antibody. The maleimide-derivative ofPEG-PE is included in the liposome preparation as described above andbelow and the antibody can be conjugated with the liposome via thesulfhydryl group at pH 7.2.

Amine modifications making use of cross-linking agents such as EDC aretaught in Endoh et al. 1981 J. Immun. Meth. 44:79-85; Dunnick 1975 J.Nuclear. Med. 16:483-487; Alternatively, direct modification ofantibodies with activated fatty acids, such as N-hydroxysuccinimide(NHS) eater or palmitic acid, prior to incorporation into a liposomemembrane, typically by detergent dialysis procedures (Huang et al. 1980,J. Biol. Chem. 255:8015-8018. Reagents, such as EDC, are used inconjunction with NHS to activate acidic functions on liposomes, whichare then conjugated to the amino groups on antibodies. Better control ofthe conjugation reaction can be achieved using heterobifunctionalcross-linkers which efficiently introduce a unique and selectivereactive function, such as a protected thiol or maleimide group.Examples of these crosslinkers are SPDP (Barbet et al. 1981 J.Supramolec. Struct. Cell. Biochem. 16:243-258), S-acetylthioglycolicacid N-hydroxysuccinimide ester (SATA, Jones 1993 Biochim. Biophys.Acta. 1152:231-32; Schwendener 1990 Biochim. Biophys. Acta. 1026:69-79and 4-(p-maleimidophenyl)butyric acid N-hydroxysuccinimide ester (SMPB(Hansen 1995 Biochim. Biophys. Acta. 1239:133-144). Antibodies whichhave been activated by these crosslinkers can, after deprotection whereappropriate, react with activated lipids in liposome bilayers. Maleimideand protected thiol-derivatized lipids are available from commercialsources for this purpose.

Deprotection of 3-pyridyl disulfides is usually effected by DTT andoccasionally by sonic other mercaptan. Once deprotected, sulfhydrylgroups can react with maleimide (for example SMPB-modified conjugates)or iodo (for example, iodoacetic acid N-hydroxysuccinimide ester(SIAA)-modified conjugates) groups. Maleimide groups are recommendedsince iodo functions can react with amino groups in either of thesubstrates, leading to undesirable side products. Deprotection is notrequired for these reagents.

Indirect Conjugation Methods:

Biotin-avidin—For example, a biotin conjugated antibody may be bound toa particle (e.g., liposome) containing a streptavidin. Alternatively,the biotinylated antibody may be conjugated to a biotin derivatizedliposome by an avidin or streptavidin linker. Ahmad et al., Cancer Res.,52: 4817-4820 (1992) which is herein incorporated by reference,describes such a mode of coupling. When monovalent Fab molecules areused, typically about 30 to 125 and more typically about 50 to 100 Fab′molecules per liposome are used.

Binding via protein A/G/L-liposome conjugates targeted to the Fc chainof antibodies is taught in Matthay et al. 1986 Cancer Res. 46:4904-4910;Machy et al. 1983 Biochem. Biophys. Acta. 901:157-160.

Loading of the particle with the pharmaceutical agent can be effectedconcomitant with, or following particle assembly.

Thus, in one preferred embodiment, for example, when the pharmaceuticalagent is a nucleic acid, e.g., DNA, RNA, siRNA, plasmid DNA,short-hairpin RNA, small temporal RNA (stRNA), microRNA (miRNA), RNAmimetics, or heterochromatic siRNA, the nucleic acid agent of interesthas a charged backbone that prevents efficient encapsulation in thelipid particle. Accordingly, the nucleic acid agent of interest may becondensed with a cationic polymer, e.g., PEI, polyamine spermidine, andspermine, or cationic peptide, e.g., protamine and polylysine, prior toencapsulation in the lipid particle. In one embodiment, the agent is notcondensed with a cationic polymer.

In another embodiment, the agent of interest is encapsulated in thelipid particle in the following manner. The particle is providedlyophilized. The agent of interest is in an aqueous solution. The agentof interest in aqueous solution is utilized to rehydrate the lyophilizedlipid particle. Thus, the agent of interest is encapsulated in therehydrated lipid particle.

In one embodiment, two agents of interest may be delivered by theparticles (e.g., lipid based particle). One agent is hydrophobic and theother is hydrophilic. The hydrophobic agent may be added to the lipidparticle during formation of the lipid particle. The hydrophobic agentassociates with the lipid portion of the lipid particle. The hydrophilicagent is added in the is aqueous solution rehydrating the lyophilizedlipid particle. In an exemplary embodiment of two agent delivery acondensed siRNA is encapsulated in a liposome and wherein a drug that ispoorly soluble in aqueous solution is associated with the lipid portionof the lipid particle. As used herein, “poorly soluble in aqueoussolution” refers to a composition that is less that 10% soluble inwater.

As used herein “loading” refers to encapsulating or absorbing.

The term “encapsulated” as used herein refers to the pharmaceuticalagent being distributed in the interior portion of the particles.Preferably, the pharmaceutical agents are homogenously distributed.Homogeneous distribution of a pharmaceutical agent in polymer particlesis known as a matrix encapsulation. However, due to the manufacturingprocess it is foreseen that minor amounts of the pharmaceutical agentmay also be present on the outside of the particle and/or mixed with thepolymer making up the shell of the particle.

As used herein “absorbed” refers to binding of the pharmaceutical agentto the outer surface of the particle.

Since pharmaceutical agents described herein (e.g. cytotoxic agents) areattached to affinity moieties which target senescent cells, the presentinventors propose that these complexes may be used to treat subjectshaving diseases associated with cell senescence.

As used herein, the term “subject” refers to a mammalian subject,preferably a human.

A number of diseases and conditions, which involve an inflammatoryresponse can be treated using the methodology described hereinabove.Examples of such diseases and conditions are summarized infra.

Inflammatory diseases—Include, but are not limited to, chronicinflammatory diseases and acute inflammatory diseases.

Inflammatory Diseases

Examples of inflammatory diseases include, but are not limited to, thediseases listed below.

An inflammatory disease, such as asthma.

Inflammatory diseases include, but are not limited to, rheumatoiddiseases, rheumatoid autoimmune diseases, rheumatoid arthritis (Krenn V.et al., Histol Histopathol 2000 July; 15 (3):791), spondylitis,ankylosing spondylitis (Jan Voswinkel et al., Arthritis Res 2001; 3 (3):189), systemic diseases, systemic autoimmune diseases, systemic lupuserythematosus (Erikson J. et al., Immunol Res 1998; 17 (1-2):49),sclerosis, systemic sclerosis (Renaudineau Y. et al., Clin Diagn LabImmunol. 1999 March; 6 (2):156); Chan O T. et al., Immunol Rev 1999June; 169:107), glandular diseases, glandular autoimmune diseases,pancreatic autoimmune diseases, diabetes, Type I diabetes (Zimmet P.Diabetes Res Clin Pract 1996 October; 34 Suppl:S125), thyroid diseases,autoimmune thyroid diseases, Graves' disease (Orgiazzi J. EndocrinolMetab Clin North Am 2000 June; 29 (2):339), thyroiditis, spontaneousautoimmune thyroiditis (Braley-Mullen H. and Yu S, J Immunol 2000 Dec.15; 165 (12):7262), Hashimoto's thyroiditis (Toyoda N. et al., NipponRinsho 1999 August; 57 (8):1810), myxedema, idiopathic myxedema (MitsumaT. Nippon Rinsho. 1999 August; 57 (8):1759), autoimmune reproductivediseases, ovarian diseases, ovarian autoimmunity (Garza K M, et al., JReprod Immunol 1998 February; 37 (2):87), autoimmune anti-sperminfertility (Diekman A B. et al., Am J Reprod Immunol. 2000 March; 43(3):134), repeated fetal loss (Tincani A. et al., Lupus 1998; 7 Suppl2:S107-9), neurodegenerative diseases, neurological diseases,neurological autoimmune diseases, multiple sclerosis (Cross A H. et al.,J Neuroimmunol 2001 Jan. 1; 112 (1-2):1), Alzheimer's disease (Oron L.et al., J Neural Transm Suppl. 1997; 49:77), myasthenia gravis (InfanteA J. And Kraig E, Int Rev Immunol 1999; 18 (1-2):83), motor neuropathies(Kornberg A J. J Clin Neurosci. 2000 May; 7 (3):191), Guillain-Barresyndrome, neuropathies and autoimmune neuropathies (Kusunoki S. Am J MedSci. 2000 April; 319 (4):234), myasthenic diseases, Lambert-Eatonmyasthenic syndrome (Takamori M. Am J Med Sci. 2000 April; 319 (4):204),paraneoplastic neurological diseases, cerebellar atrophy, paraneoplasticcerebellar atrophy, non-paraneoplastic stiff man syndrome, cerebellaratrophies, progressive cerebellar atrophies, encephalitis, Rasmussen'sencephalitis, amyotrophic lateral sclerosis, Sydeham chorea, Gilles dela Tourette syndrome, polyendocrinopathies, autoimmunepolyendocrinopathies (Antoine J C. and Honnorat J. Rev Neurol (Paris)2000 January; 156 (1):23); neuropathies, dysimmune neuropathies(Nobile-Orazio E. et al., Electroencephalogr Clin Neurophysiol Suppl1999; 50:419); neuromyotonia, acquired neuromyotonia, arthrogryposismultiplex congenita (Vincent A. et al., Ann N Y Acad. Sci. 1998 May 13;841:482), cardiovascular diseases, cardiovascular autoimmune diseases,atherosclerosis (Matsuura E. et al., Lupus. 1998; 7 Suppl 2:S135),myocardial infarction (Vaarala O. Lupus. 1998; 7 Suppl 2:S132),thrombosis (Tincani A. et al., Lupus 1998; 7 Suppl 2:S107-9),granulomatosis, Wegener's granulomatosis, arteritis, Takayasu'sarteritis and Kawasaki syndrome (Praprotnik S. et al., Wien KlinWochenschr 2000 Aug. 25; 112 (15-16):660); anti-factor VIII autoimmunedisease (Lacroix-Desmazes S. et al., Semin Thromb Hemost. 2000; 26(2):157); vasculitises, necrotizing small vessel vascullitises,microscopic polyangiitis, Churg and Strauss syndrome,glomerulonephritis, pauci-immune focal necrotizing glomerulonephritis,crescentic glomerulonephritis (Noel L H. Ann Med Interne (Paris). 2000May; 151 (3):178); antiphospholipid syndrome (Hamholz R. et al., J ClinApheresis 1999; 14 (4):171); heart failure, agonist-likebeta-adrenoceptor antibodies in heart failure (Wallukat G. et al., Am JCardiol. 1999 Jun. 17; 83 (12A):75H), thrombocytopenic purpura (MocciaF. Ann Ital Med Int. 1999 April-June; 14 (2):114); hemolytic anemia,autoimmune hemolytic anemia (Efremov D G. et al., Leuk Lymphoma 1998January; 28 (3-4):285), gastrointestinal diseases, autoimmune diseasesof the gastrointestinal tract, intestinal diseases, chronic inflammatoryintestinal disease (Garcia Herola A. et al., Gastroenterol Hepatol. 2000January; 23 (1):16), celiac disease (Landau Y E. and Shoenfeld Y.Harefuah 2000 Jan. 16; 138 (2):122), autoimmune diseases of themusculature, myositis, autoimmune myositis, Sjogren's syndrome (Feist E.et al., Int Arch Allergy Immunol 2000 September; 123 (1):92); smoothmuscle autoimmune disease (Zauli D. et al., Biomed Pharmacother 1999June; 53 (5-6):234), hepatic diseases, hepatic autoimmune diseases,autoimmune hepatitis (Manns M P. J Hepatol 2000 August; 33 (2):326) andprimary biliary cirrhosis (Strassburg C P. et al., Eur J GastroenterolHepatol. 1999 June; 11 (6):595).

Further inflammatory diseases include, but are not limited to,rheumatoid diseases, rheumatoid arthritis (Tisch R, McDevitt H O. ProcNatl Acad Sci USA 1994 Jan. 18; 91 (2):437), systemic diseases, systemicautoimmune diseases, systemic lupus erythematosus (Datta S K., Lupus1998; 7 (9):591), glandular diseases, glandular autoimmune diseases,pancreatic diseases, pancreatic autoimmune diseases, Type 1 diabetes(Castano L. and Eisenbarth G S. Ann. Rev. Immunol. 8:647); thyroiddiseases, autoimmune thyroid diseases, Graves' disease (Sakata S. etal., Mol Cell Endocrinol 1993 March; 92 (1):77); ovarian diseases (GarzaK M. et al., J Reprod Immunol 1998 February; 37 (2):87), prostatitis,autoimmune prostatitis (Alexander R B. et al., Urology 1997 December; 50(6):893), polyglandular syndrome, autoimmune polyglandular syndrome,Type I autoimmune polyglandular syndrome (Hara T. et al., Blood. 1991Mar. 1; 77 (5):1127), neurological diseases, autoimmune neurologicaldiseases, multiple sclerosis, neuritis, optic neuritis (Soderstrom M. etal., J Neurol Neurosurg Psychiatry 1994 May; 57 (5):544), myastheniagravis (Oshima. M. et al., Eur J Immunol 1990 December; 20 (12):2563),stiff-man syndrome (Hiemstra H S. et al., Proc Natl Acad Sci USA 2001Mar. 27; 98 (7):3988), cardiovascular diseases, cardiac autoimmunity inChagas' disease (Cunha-Neto E. et al., J Clin Invest 1996 Oct. 15; 98(8):1709), autoimmune thrombocytopenic purpura (Semple J W. et al.,Blood 1996 May 15; 87 (10):4245), anti-helper T lymphocyte autoimmunity(Caporossi A P. et al., Viral Immunol 1998; 11 (1):9), hemolytic anemia(Sallah S. et al., Ann Hematol 1997 March; 74 (3):139), hepaticdiseases, hepatic autoimmune diseases, hepatitis, chronic activehepatitis (Franco A. et al., Clin Immunol Immunopathol 1990 March; 54(3):382), biliary cirrhosis, primary biliary cirrhosis (Jones D E. ClinSci (Colch) 1996 November; 91 (5):551), nephric diseases, nephricautoimmune diseases, nephritis, interstitial nephritis (Kelly C J. J AmSoc Nephrol 1990 August; 1 (2):140), connective tissue diseases, eardiseases, autoimmune connective tissue diseases, autoimmune ear disease(Yoo T J. et al., Cell Immunol 1994 August; 157 (1):249), disease of theinner ear (Gloddek B. et al., Ann N Y Acad Sci 1997 Dec. 29; 830:266),skin diseases, cutaneous diseases, dermal diseases, bullous skindiseases, pemphigus vulgaris, bullous pemphigoid and pemphigusfoliaceus.

Examples of inflammatory diseases include, but are not limited to,contact dermatitis and drug eruption.

Examples of types of T lymphocyte mediating inflammatory diseasesinclude, but are not limited to, helper T lymphocytes and cytotoxic Tlymphocytes.

Examples of helper T lymphocyte-mediated inflammatory diseases include,but are not limited to, T_(h)1 lymphocyte mediated inflammation andT_(h)2 lymphocyte mediated inflammation.

Autoimmune Diseases

Include, but are not limited to, cardiovascular diseases, rheumatoiddiseases, glandular diseases, gastrointestinal diseases, cutaneousdiseases, hepatic diseases, neurological diseases, muscular diseases,nephric diseases, diseases related to reproduction, connective tissuediseases and systemic diseases.

Examples of autoimmune cardiovascular diseases include, but are notlimited to atherosclerosis (Matsuura E. et al., Lupus. 1998; 7 Suppl2:S135), myocardial infarction (Vaarala O. Lupus. 1998; 7 Suppl 2:S132),thrombosis (Tincani A. et al., Lupus 1998; 7 Suppl 2:S107-9), Wegener'sgranulomatosis, Takayasu's arteritis, Kawasaki syndrome (Praprotnik S.et al., Wien Klin Wochenschr 2000 Aug. 25; 112 (15-16):660), anti-factorVIII autoimmune disease (Lacroix-Desmazes S. et al., Semin ThrombHemost. 2000; 26 (2):157), necrotizing small vessel vasculitis,microscopic polyangiitis, Churg and Strauss syndrome, pauci-immune focalnecrotizing and crescentic glomerulonephritis (Noel L H. Ann Med Interne(Paris). 2000 May; 151 (3):178), antiphospholipid syndrome (Flamholz R.et al., J Clin Apheresis 1999; 14 (4):171), antibody-induced heartfailure (Wallukat G. et al., Am J Cardiol. 1999 Jun. 17; 83 (12A):75H),thrombocytopenic purpura (Moccia F. Ann Ital Med. Int. 1999 April-June;14 (2):114; Semple J W. et al., Blood 1996 May 15; 87 (10):4245),autoimmune hemolytic anemia (Efremov D G. et al., Leuk Lymphoma 1998January; 28 (3-4):285; Sallah S. et al., Ann Hematol 1997 March; 74(3):139), cardiac autoimmunity in Chagas' disease (Cunha-Neto E. et al.,J Clin Invest 1996 Oct. 15; 98 (8):1709) and anti-helper T lymphocyteautoimmunity (Caporossi A P. et al., Viral Immunol 1998; 11 (1):9).

Examples of autoimmune rheumatoid diseases include, but are not limitedto rheumatoid arthritis (Krenn V. et al., Histol Histopathol 2000 July;15 (3):791; Tisch R, McDevitt H O. Proc Natl Acad Sci units S A 1994Jan. 18; 91 (2):437) and ankylosing spondylitis (Jan Voswinkel et al.,Arthritis Res 2001; 3 (3): 189).

Examples of autoimmune glandular diseases include, but are not limitedto, pancreatic disease, Type I diabetes, thyroid disease, Graves'disease, thyroiditis, spontaneous autoimmune thyroiditis, Hashimoto'sthyroiditis, idiopathic myxedema, ovarian autoimmunity, autoimmuneanti-sperm infertility, autoimmune prostatitis and Type I autoimmunepolyglandular syndrome. Diseases include, but are not limited toautoimmune diseases of the pancreas, Type 1 diabetes (Castano L. andEisenbarth G S. Ann. Rev. Immunol. 8:647; Zimmet P. Diabetes Res ClinPract 1996 October; 34 Suppl:S125), autoimmune thyroid diseases, Graves'disease (Orgiazzi J. Endocrinol Metab Clin North Am 2000 June; 29(2):339; Sakata S. et al., Mol Cell Endocrinol 1993 March; 92 (1):77),spontaneous autoimmune thyroiditis (Braley-Mullen H. and Yu S, J Immunol2000 Dec. 15; 165 (12):7262), Hashimoto's thyroiditis (Toyoda N. et al.,Nippon Rinsho 1999 August; 57 (8):1810), idiopathic myxedema (Mitsuma T.Nippon Rinsho. 1999 August; 57 (8):1759), ovarian autoimmunity (Garza KM. et al., J Reprod Immunol 1998 February; 37 (2):87), autoimmuneanti-sperm infertility (Diekman A B. et al., Am J Reprod Immunol. 2000March; 43 (3):134), autoimmune prostatitis (Alexander R B. et al.,Urology 1997 December; 50 (6):893) and Type I autoimmune polyglandularsyndrome (Mara T. et al., Blood. 1991 Mar. 1; 77 (5):1127).

Examples of autoimmune gastrointestinal diseases include, but are notlimited to, chronic inflammatory intestinal diseases (Garcia Herola A.et al., Gastroenterol Hepatol. 2000 January; 23 (1):16), celiac disease(Landau Y E. and Shoenfeld Y. Harefuah 2000 Jan. 16; 138 (2):122),colitis, ileitis and Crohn's disease.

Examples of autoimmune cutaneous diseases include, but are not limitedto, autoimmune bullous skin diseases, such as, but are not limited to,pemphigus vulgaris, bullous pemphigoid and pemphigus foliaceus.

Examples of autoimmune hepatic diseases include, but are not limited to,hepatitis, autoimmune chronic active hepatitis (Franco A. et al., ClinImmunol Immunopathol 1990 March; 54 (3):382), primary biliary cirrhosis(Jones D E. Clin Sci (Colch) 1996 November; 91 (5):551; Strassburg C P.et al., Eur J Gastroenterol Hepatol. 1999 June; 11 (6):595) andautoimmune hepatitis (Manns M P. J Hepatol 2000 August; 33 (2):326).

Examples of autoimmune neurological diseases include, but are notlimited to, multiple sclerosis (Cross A H. et al., J Neuroimmunol 2001Jan. 1; 112 (1-2):1), Alzheimer's disease (Oron L. et al., J NeuralTransm Suppl. 1997; 49:77), myasthenia gravis (Infante A J. And Kraig E,Int Rev Immunol. 1999; 18 (1-2):83; Oshima M. et al., Eur J Immunol 1990December; 20 (12):2563), neuropathies, motor neuropathies (Kornberg A J.J Clin Neurosci. 2000 May; 7 (3):191); Guillain-Barre syndrome andautoimmune neuropathies (Kusunoki S. Am J Med Sci. 2000 April; 319(4):234), myasthenia, Lambert-Eaton myasthenic syndrome (Takamori M. AmJ Med Sci. 2000 April; 319 (4):204); paraneoplastic neurologicaldiseases, cerebellar atrophy, paraneoplastic cerebellar atrophy andstiff-man syndrome (Hiemstra H S. et al., Proc Natl Acad Sci units S A2001 Mar. 27; 98 (7):3988); non-paraneoplastic stiff man syndrome,progressive cerebellar atrophies, encephalitis, Rasmussen'sencephalitis, amyotrophic lateral sclerosis, Sydeham chorea, Gilles dela Tourette syndrome and autoimmune polyendocrinopathies (Antoine J C.and Honnorat J. Rev Neurol (Paris) 2000 January; 156 (1):23); dysimmuneneuropathies (Nubile-Orazio E. et al., Electroencephalogr ClinNeurophysiol Suppl 1999; 50:419); acquired neuromyotonia, arthrogryposismultiplex congenita (Vincent A. et al., Ann N Y Acad Sci. 1998 May 13;841:482), neuritis, optic neuritis (Soderstrom M. et al., J NeurolNeurosurg Psychiatry 1994 May; 57 (5):544) and neurodegenerativediseases.

Examples of autoimmune muscular diseases include, but are not limitedto, myositis, autoimmune myositis and primary Sjogren's syndrome (FeistE. et al., Int Arch Allergy Immunol 2000 September; 123 (1):92) andsmooth muscle autoimmune disease (Zauli D. et al., Biomed Pharmacother1999 June; 53 (5-6):234).

Examples of autoimmune nephric diseases include, but are not limited to,nephritis and autoimmune interstitial nephritis (Kelly C J. J Am SocNephrol 1990 August; 1 (2):140).

Examples of autoimmune diseases related to reproduction include, but arenot limited to, repeated fetal loss (Tincani A. et al., Lupus 1998; 7Suppl 2:S107-9).

Examples of autoimmune connective tissue diseases include, but are notlimited to, ear diseases, autoimmune ear diseases (Yoo T J. et al., CellImmunol 1994 August; 157 (1):249) and autoimmune diseases of the innerear (Gloddek B. et al., Ann N Y Acad Sci 1997 Dec. 29; 830:266).

Examples of autoimmune systemic diseases include, but are not limitedto, systemic, lupus erythematosus (Erikson J. et al., Immunol Res 1998;17 (1-2):49) and systemic sclerosis (Renaudineau Y. et al., Clin DiagnLab Immunol. 1999 March; 6 (2):156); Chan O T. et al., Immunol Rev 1999June; 169:107).

Infectious Diseases

Examples of infectious diseases include, but are not limited to, chronicinfectious diseases, subacute infectious diseases, acute infectiousdiseases, viral diseases, bacterial diseases, protozoan diseases,parasitic diseases, fungal diseases, mycoplasma diseases and priondiseases.

Graft Rejection Diseases

Examples of diseases associated with transplantation of a graft include,but are not limited to, graft rejection, chronic graft rejection,subacute graft rejection, hyperacute graft rejection, acute graftrejection and graft versus host disease.

Allergic Diseases

Examples of allergic diseases include, but are not limited to, asthma,hives, urticaria, pollen allergy, dust mite allergy, venom allergy,cosmetics allergy, latex allergy, chemical allergy, drug allergy, insectbite allergy, animal dander allergy, stinging plant allergy, poison ivyallergy and food allergy.

According to a particular embodiment, the agents (and combinationsthereof) are used to treat pre-malignant lesions.

As used herein, the phrase “pre-malignant lesion” refers to a mass ofcells and/or tissue having increased probability of transforming into amalignant tumor. Examples of pre-malignant lesions include, but are notlimited to, adenomatous polyps, Barrett's esophagus, PancreaticIntraepithelial Neoplasia (PanIN), IPMN (Intraductal Papillary MucinusNeoplasia), DCIS (Ductal Carcinoma in Situ) in the breast, leukoplakiaand erythroplakia. Thus, the pre-malignant lesion which is treated usingthe agents of this aspect of the present invention can transform into amalignant solid or non-solid (e.g., hematological malignancies) cancer(or tumor). According to a particular embodiment, the pre-malignantlesion which is treated using the agents of the present invention is anadenomatous polyp of the colon, an adenomatous polyp of the rectum, anadenomatous polyp of the small bowel and Barrett's esophagus.

Examples of fibrotic diseases include diseases of an epithelial barriertissue, diseases of the skin, lung or gut.

Contemplated fibrotic diseases which may be treated using the agentsdescribed herein include but are not limited to eosinophilicesophagitis, hypereosinophilic syndromes (HES), Loeffler'sendomyocarditis, endomyocardial fibrosis, idiopathic pulmonary fibrosis,and scleroderma.

According to a particular embodiment the agents are used for treatingliver fibrosis, wound healing, skin fibrosis, pulmonary disease, kidneyfibrosis, prostatitis, atherosclerosis, arthritis, osteoporosis orpancreatitis.

An exemplary pulmonary disease contemplated by the present invention ischronic obstructive pulmonary disease (COPD) or Idiopathic pulmonaryfibrosis.

According to sill another embodiment, the disease is associated withcartilage degeneration—e.g. arthritis.

According to still another embodiment, the disease is associated withbone degeneration—e.g. osteoporosis.

According to still another embodiment, the disease is not cancer.

The complexes of the present invention may be provided per se or may beformulated in compositions intended for a particular use.

Since the complexes of the present invention selectively targetsenescent cells, the present inventors contemplate that another usethereof is in cosmetic compositions as anti-aging agents forrejuvenating the skin. Thus, the agents of the present invention may beformulated for cosmetics.

Such compositions typically comprise pharmaceutically acceptableexcipient, notably dermatologically acceptable suitable for externaltopical application.

The cosmetic composition according to the present invention may furthercomprise at least one pharmaceutical adjuvant known to the personskilled in the art, selected from thickeners, preservatives, fragrances,colorants, chemical or mineral filters, moisturizing agents, thermalspring water, etc.

The composition may comprise at least one agent selected from asebum-regulating agent, an antibacterial agent, an antifungal agent, akeratolytic agent, a keratoregulating agent, an astringent, ananti-inflammatory/anti-irritant, an antioxidant/free-radical scavenger,a cicatrizing agent, an anti-aging agent and/or a moisturizing agent.

The term “sebum-regulating agent” refers, for example, to 5-α-reductaseinhibitors, notably the active agent 5-α-Avocuta® sold by LaboratoiresExpanscience. Zinc and gluconate salts thereof, salicylate andpyroglutamic acid, also have sebum-suppressing activity. Mention mayalso be made of spironolactone, an anti-androgen and aldosteroneantagonist, which significantly reduces the sebum secretion rate after12 weeks of application. Other extracted molecules, for example fromseeds of the pumpkin Cucurbita pepo, and squash seed oil, as well aspalm cabbage, limit sebum production by inhibiting 5-α-reductasetranscription and activity. Other sebum-regulating agents of lipidorigin that act on sebum quality, such as linoleic acid, are ofinterest.

The terms “anti-bacterial agent” and “antifungal agent” refer tomolecules that limit the growth of or destroy pathogenic microorganismssuch as certain bacteria like P. acnes or certain fungi (Malasseziafurfur). The most traditional are preservatives generally used incosmetics or nutraceuticals, molecules with anti-bacterial activity(pseudo-preservatives) such as caprylic derivatives (capryloyl glycine,glyceryl caprylate, etc.), such as hexanediol and sodium levulinate,zinc and copper derivatives (gluconate and PCA), phytosphingosine andderivatives thereof, benzoyl peroxide, piroctone olamine, zincpyrithione, selenium sulfide, econazole, ketoconazole, or localantibiotics such as erythromycin and clindamycin, etc.

The terms “keratoregulating agent” and “keratolytic agent” refer to anagent that regulates or helps the elimination of dead cells of thestratum corneum of the epidermis. The most commonly usedkeratoregulating/keratolytic agents include: alpha-hydroxy acids (AHAs)of fruits (citric acid, glycolic acid, malic acid, lactic acid, etc.),AHA esters, combinations of AHAs with other molecules such as thecombination of malic acid and almond proteins (Keratolite®), thecombination of glycolic acid or lactic acid with arginine or thecombination of hydroxy acid with lipid molecules such as LHA®(lipo-hydroxy acid), amphoteric hydroxy acid complexes (AHCare), willowbark (Salix alba bark extract), azelaic acid and salts and estersthereof, salicylic acid and derivatives thereof such as capryloylsalicylic acid or in combination with other molecules such as thecombination of salicylic acid and polysaccharide (beta-hydroxy acid, orBHA), tazarotene, adapalene, as well as molecules of the retinoid familysuch as tretinoin, retinaldehyde, isotretinoin and retinol.

The term “astringent” refers to an agent that helps constrict pores, themost commonly used being polyphenols, zinc derivatives and witch hazel.

The term “anti-inflammatory/anti-irritant” refers to an agent thatlimits the inflammatory reaction led by cytokines or arachidonic acidmetabolism mediators and has soothing and anti-irritating properties.The most traditional are glycyrrhetinic acid (licorice derivative) andsalts and esters thereof, alpha-bisabolol, Ginkgo biloba, Calendula,lipoic acid, beta-carotene, vitamin B3 (niacinamide, nicotinamide),vitamin E, vitamin C, vitamin B12, flavonoids (green tea, quercetin,etc.), lycopene or lutein, avocado sugars, avocado oleodistillate,arabinogalactan, lupin peptides, lupin total extract, quinoa peptideextract, Cycloceramide® (oxazoline derivative), anti-glycation agentssuch as carnosine, N-acetyl-cysteine, isoflavones such as, for example,genistein/genistin, daidzein/daidzin, spring water or thermal springwater (eau d'Avene, eau de la Roche Posay, eau de Saint Gervais, eaud'Uriage, eau de Gamarde), goji extracts (Lycium barbarum), plant aminoacid peptides or complexes, topical dapsone, or anti-inflammatory drugs.

The term “antioxidant” refers to a molecule that decreases or preventsthe oxidation of other chemical substances. Theantioxidants/tree-radical scavengers that may be used in combination areadvantageously selected from the group comprised of thiols and phenols,licorice derivatives such as glycyrrhetinic acid and salts and estersthereof, alpha-bisabolol, Ginkgo biloba extract, Calendula extract,Cycloceramide® (oxazoline derivative), avocado peptides, trace elementssuch as copper, zinc and selenium, lipoic acid, vitamin B12, vitamin B3(niacinamide, nicotinamide), vitamin C, vitamin E, coenzyme Q10, krill,glutathione, butylated. hydroxytoluene (BHT), butylated hydroxyanisole(BHA), lycopene or lutein, beta-carotene, the family of polyphenols suchas tannins, phenolic acids, anthocyanins, flavonoids such as, forexample, extracts of green tea, of red berries, of cocoa, of grapes, ofPassiflora incamata or of Citrus, or isoflavones such as, for example,genistein/genistin and daidzein/daidzin. The group of antioxidantsfurther includes anti-glycation agents such as carnosine or certainpeptides, N-acetyl-cysteine, as well as antioxidant or free-radicalscavenging enzymes such as superoxide dismutase (SOD), catalase,glutathione peroxidase, thioredoxin reductase and agonists thereof.

The agents that cicatrize/repair the barrier function which may be usedin combination are advantageously vitamin A, panthenol (vitamin B5),Avocadofurane®, avocado sugars, lupeol, maca peptide extract, quinoapeptide extract, arabinogalactan, zinc oxide, magnesium, silicon,madecassic or asiatic acid, dextran sulfate, coenzyme Q10, glucosamineand derivatives thereof, chondroitin sulfate and on the wholeglycosaminoglycans (GAGs), dextran sulfate, ceramides, cholesterol,squalane, phospholipids, fermented or unfermented soya peptides, plantpeptides, marine, plant or biotechnological polysaccharides such asalgae extracts or fern extracts, trace elements, extracts of tannin-richplants such as tannins derived from gallic acid called gallic orhydrolysable tannins, initially found in oak gall, and catechin tanninsresulting from the polymerization of flavin units whose model isprovided by the catechu (Acacia catechu). The trace elements that may beused are advantageously selected from the group comprised of copper,magnesium, manganese, chromium, selenium, silicon, zinc and mixturesthereof.

Anti-aging agents that can act in combination to treat acne in maturesubjects are antioxidants and in particular vitamin C, vitamin A,retinol, retinal, hyaluronic acid of any molecular weight,Avocadofurane®, lupin peptides and maca peptide extract.

The most commonly used moisturizers/emollients are glycerin orderivatives thereof, urea, pyrrolidone carboxylic acid and derivativesthereof, hyaluronic acid of any molecular weight, glycosaminoglycans andany other polysaccharides of marine, plant or biotechnological originsuch as, for example, xanthan gum, Fucogel®, certain fatty acids such aslauric acid, myristic acid, monounsaturated and polyunsaturated omega-3,-6, -7 and -9 fatty acids (linoleic acid, palmitoleic acid, etc.),sunflower oleodistillate, avocado peptides and cupuacu butter.

For treatment of diseases, the agents of the present invention may beformulated in pharmaceutical compositions.

As used herein a “pharmaceutical composition” refers to a preparation ofone or more of the active ingredients described herein with otherchemical components such as physiologically suitable carriers andexcipients. The purpose of a pharmaceutical composition is to facilitateadministration of a compound to an organism.

Herein the term “active ingredient” refers to the therapeutic agent (asdescribed herein above) accountable for the biological effect which isattached directly or indirectly to the targeting agent. It will beappreciated that the pharmaceutical compositions may comprise additionalactive agents known to be useful in treating a particular disease. Thus,for example for treatment of skin fibrotic diseases, the presentinventors contemplate pharmaceutical compositions comprising the abovedescribed agents together with at least one sebum-regulating agent, anantibacterial agent, an antifungal agent, a keratolytic agent, akeratoregulating agent, an astringent, ananti-inflammatory/anti-irritant, an antioxidant/free-radical scavenger,a cicatrizing agent, an anti-aging agent and/or a moisturizing agent, asdescribed herein above.

Hereinafter, the phrases “physiologically acceptable carrier” and“pharmaceutically acceptable carrier” which may be interchangeably usedrefer to a carrier or a diluent that does not cause significantirritation to an organism and does not abrogate the biological activityand properties of the administered compound. An adjuvant is includedunder these phrases.

Herein the term “excipient” refers to an inert substance added to apharmaceutical composition to further facilitate administration of anactive ingredient. Examples, without limitation, of excipients includecalcium carbonate, calcium phosphate, various sugars and types ofstarch, cellulose derivatives, gelatin, vegetable oils and polyethyleneglycols.

Pharmaceutical compositions of the present invention may be manufacturedby processes well known in the art, e.g., by means of conventionalmixing, dissolving, granulating, dragee-making, levigating, emulsifying,encapsulating, entrapping or lyophilizing processes.

Pharmaceutical compositions for use in accordance with the presentinvention thus may be formulated in conventional manner using one ormore physiologically acceptable carriers comprising excipients andauxiliaries, which facilitate processing of the active ingredients intopreparations which, can be used pharmaceutically. Proper formulation isdependent upon the route of administration chosen.

For injection, the active ingredients of the pharmaceutical compositionmay be formulated in aqueous solutions, preferably in physiologicallycompatible buffers such as Hank's solution, Ringer's solution, orphysiological salt buffer.

Suitable routes of administration may, for example, include oral,rectal, transmucosal, especially transnasal, intestinal or parenteraldelivery, including intramuscular, subcutaneous and intramedullaryinjections as well as intrathecal, direct intraventricular,intracardiac, e.g., into the right or left ventricular cavity, into thecommon coronary artery, intravenous, intraperitoneal, intranasal, orintraocular injections.

According to a particular embodiment, the route of administration is viatopical delivery.

Alternately, one may administer the pharmaceutical composition in alocal rather than systemic manner, for example, via injection of thepharmaceutical composition directly into a tissue region of a patient.

Pharmaceutical compositions of the present invention may be manufacturedby processes well known in the art, e.g., by means of conventionalmixing, dissolving, granulating, dragee-making, levigating, emulsifying,encapsulating, entrapping or lyophilizing processes.

Pharmaceutical compositions for use in accordance with the presentinvention thus may be formulated in conventional manner using one ormore physiologically acceptable carriers comprising excipients andauxiliaries, which facilitate processing of the active ingredients intopreparations which, can be used pharmaceutically. Proper formulation isdependent upon the route of administration chosen.

For injection, the active ingredients of the pharmaceutical compositionmay be formulated in aqueous solutions, preferably in physiologicallycompatible buffers such as Hank's solution, Ringer's solution, orphysiological salt buffer. For transmucosal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art.

For oral administration, the pharmaceutical composition can beformulated readily by combining the active compounds withpharmaceutically acceptable carriers well known in the art. Suchcarriers enable the pharmaceutical composition to be formulated astablets, pills, dragees, capsules, liquids, gels, syrups, slurries,suspensions, and the like, for oral ingestion by a patient.Pharmacological preparations for oral use can be made using a solidexcipient, optionally grinding the resulting mixture, and processing themixture of granules, after adding suitable auxiliaries if desired, toobtain tablets or dragee cores. Suitable excipients are, in particular,fillers such as sugars, including lactose, sucrose, mannitol, orsorbitol; cellulose preparations such as, for example, maize starch,wheat starch, rice starch, potato starch, gelatin, gum tragacanth,methyl cellulose, hydroxypropylmethyl-cellulose, sodiumcarbomethylcellulose; and/or physiologically acceptable polymers such aspolyvinylpyrrolidone (PVP). If desired, disintegrating agents may beadded, such as cross-linked polyvinyl pyrrolidone, agar, or alginic acidor a salt thereof such as sodium alginate.

Dragee cores are provided with suitable coatings. For this purpose,concentrated sugar solutions may be used which may optionally containgum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethyleneglycol, titanium dioxide, lacquer solutions and suitable organicsolvents or solvent mixtures. Dyestuffs or pigments may be added to thetablets or dragee coatings for identification or to characterizedifferent combinations of active compound doses.

Pharmaceutical compositions which can be used orally, include push-fitcapsules made of gelatin as well as soft, sealed capsules made ofgelatin and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules may contain the active ingredients in admixture with fillersuch as lactose, binders such as starches, lubricants such as talc ormagnesium stearate and, optionally, stabilizers. In soft capsules, theactive ingredients may be dissolved or suspended in suitable liquids,such as fatty oils, liquid paraffin, or liquid polyethylene glycols. Inaddition, stabilizers may be added. All formulations for oraladministration should be in dosages suitable for the chosen route ofadministration.

For buccal administration, the compositions may take the form of tabletsor lozenges formulated in conventional manner.

For administration by nasal inhalation, the active ingredients for useaccording to the present invention are conveniently delivered in theform of an aerosol spray presentation from a pressurized pack or anebulizer with the use of a suitable propellant, e.g.,dichlorodifluoromethane, trichlorofluoromethane,dichloro-tetrafluoroethane or carbon dioxide. In the case of apressurized aerosol, the dosage unit may be determined by providing avalve to deliver a metered amount. Capsules and cartridges of, e.g.,gelatin for use in a dispenser may be formulated containing a powder mixof the compound and a suitable powder base such as lactose or starch.

The pharmaceutical composition described herein may be formulated forparenteral administration, e.g., by bolus injection or continuousinfusion. Formulations for injection may be presented in unit dosageform, e.g., in ampoules or in multidose containers with optionally, anadded preservative. The compositions may be suspensions, solutions oremulsions in oily or aqueous vehicles, and may contain formulatoryagents such as suspending, stabilizing and/or dispersing agents.

Pharmaceutical compositions for parenteral administration includeaqueous solutions of the active preparation in water-soluble form.Additionally, suspensions of the active ingredients may be prepared asappropriate oily or water based injection suspensions. Suitablelipophilic solvents or vehicles include fatty oils such as sesame oil,or synthetic fatty acids esters such as ethyl oleate, triglycerides orliposomes. Aqueous injection suspensions may contain substances, whichincrease the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol or dextran. Optionally, the suspension may alsocontain suitable stabilizers or agents which increase the solubility ofthe active ingredients to allow for the preparation of highlyconcentrated solutions.

Alternatively, the active ingredient may be in powder form forconstitution with a suitable vehicle, e.g., sterile, pyrogen-free waterbased solution, before use.

The pharmaceutical composition of the present invention may also beformulated in rectal compositions such as suppositories or retentionenemas, using, e.g., conventional suppository bases such as cocoa butteror other glycerides.

Pharmaceutical compositions suitable for use in context of the presentinvention include compositions wherein the active ingredients arecontained in an amount effective to achieve the intended purpose. Morespecifically, a therapeutically effective amount means an amount ofactive ingredients (e.g, siRNA agents together with targeting agents)effective to prevent, alleviate or ameliorate symptoms of a disorder(e.g., fibrotic or inflammatory disease) or prolong the survival of thesubject being treated.

Determination of a therapeutically effective amount is well within thecapability of those skilled in the art, especially in light of thedetailed disclosure provided herein.

For any preparation used in the methods of the invention, thetherapeutically effective amount or dose can be estimated from animalmodels (e.g. mouse models of liver fibrosis induced by CCl₄, mouse modelof pancreatitis induced by Caerulein, mouse model of COPD) to achieve adesired concentration or titer. Such information can be used to moreaccurately determine useful doses in humans.

Toxicity and therapeutic efficacy of the active ingredients describedherein can be determined by standard pharmaceutical procedures inexperimental animals. The data obtained from these animal studies can beused in formulating a range of dosage for use in human. The dosage mayvary depending upon the dosage form employed and the route ofadministration utilized. The exact formulation, route of administrationand dosage can be chosen by the individual physician in view of thepatient's condition. (See e.g., Fingl, et al., 1975, in “ThePharmacological Basis of Therapeutics”, Ch. 1 p. 1).

Dosage amount and interval may be adjusted individually to provide cellnumbers sufficient to induce normoglycemia (minimal effectiveconcentration, MEC). The MEC will vary for each preparation, but can beestimated from in vitro data. Dosages necessary to achieve the MEC willdepend on individual characteristics and route of administration.Detection assays can be used to determine plasma concentrations.

The amount of a composition to be administered will, of course, bedependent on the subject being treated, the severity of the affliction,the manner of administration, the judgment of the prescribing physician,etc.

Compositions of the present invention may, if desired, be presented in apack or dispenser device, such as an FDA approved kit, which may containone or more unit dosage forms containing the active ingredient. The packmay, for example, comprise metal or plastic foil, such as a blisterpack. The pack or dispenser device may be accompanied by instructionsfor administration. The pack or dispenser may also be accommodated by anotice associated with the container in a form prescribed by agovernmental agency regulating the manufacture, use or sale ofpharmaceuticals, which notice is reflective of approval by the agency ofthe form of the compositions or human or veterinary administration. Suchnotice, for example, may be of labeling approved by the U.S. Food andDrug Administration for prescription drugs or of an approved productinsert. Compositions comprising a preparation of the inventionformulated in a compatible pharmaceutical carrier may also be prepared,placed in an appropriate container, and labeled for treatment of anindicated condition, as if further detailed above.

Since the present inventors have found that the polypeptides HSP90B1,DNAJB4, PI4K2A, DBN1, PRKCSH, SPTBN1, NPM1, ITGA3 and any of thepolypeptides listed in Table 1, herein below are selectively upregulatedon the surface of senescent cells as compared to non-senescent cells,the present inventors propose that measuring the level of such proteinsmay be used to identify senescent cells.

Thus, according to still another aspect of the present invention thereis provided a method of identifying senescent cells in a cell populationcomprising analyzing the amount of at least one polypeptide selectedfrom the group consisting of HSP90B1, DNAJB4, PI4K2A, DBN1, PRKCSH,SPTBN1, NPM1, ITGA3 and any of the polypeptides listed in Table 1 on themembrane of the cells of the cell population, wherein a level of the atleast one polypeptide above a predetermined amount is indicative ofsenescent cells.

The identifying may be part of a method of diagnosing a diseaseassociated with senescent cells as further described herein above.

The term “diagnosing” as used herein refers to determining the presenceof a disease, classifying a disease, staging a disease, determining aseverity of a disease, monitoring disease progression, forecasting anoutcome of the disease, predicting survival and/or prospects of recovery(i.e. prognosis).

The subject may be a healthy animal or human subject undergoing aroutine well-being check up. Alternatively, the subject may be at riskof having the disease (e.g., a genetically predisposed subject, asubject with medical and/or family history of cancer, a subject who hasbeen exposed to carcinogens, occupational hazard, environmental hazard]and/or a subject who exhibits suspicious clinical signs of the disease[e.g., blood in the stool or melena, unexplained pain, sweating,unexplained fever, unexplained loss of weight up to anorexia, changes inbowel habits (constipation and/or diarrhea), tenesmus (sense ofincomplete defecation, for rectal cancer specifically), anemia and/orgeneral weakness). Still alternatively, the subject may be diagnosed ashaving a disease associated with senescent cells, but the stage is beingevaluated.

Determining an expression of any of the polypeptides listed above may beeffected on the RNA or protein level as detailed below.

According to one embodiment, the determining is effected ex vivo.

According to another embodiment, the determining is effected in vivo.

Diseases which may be diagnosed are listed herein above. According to aparticular embodiment, the disease is cancer or a premalignant disease(e.g. Pancreatic Intraepithelial Neoplasia (PanIN)).

Methods of Detecting Expression of the Polypeptides on the RNA Level

When the polypeptide is expressed solely on the membrane (e.g. ITGA3)and not in other cell compartments, RNA based methods on whole cellextracts may be used. In this scenario lysed cells may be used for thedetection of the polypeptides.

Preferably, when the polypeptide is not expressed solely on the membraneand is expressed in other cell compartments, RNA based methods areperformed on membrane extracts.

In order to detect expression of the polypeptides on the RNA level,typically polynucleotide probes (e.g. oligonucleotides or primers) areused that are capable of specifically hybridizing to their RNA or cDNAgenerated therefrom.

Preferably, the oligonucleotide probes and primers utilized by thevarious hybridization techniques described hereinabove are capable ofhybridizing to their targets under stringent hybridization conditions.

By way of example, hybridization of short nucleic acids (below 200 bp inlength, e.g. 17-40 bp in length) can be effected by the followinghybridization protocols depending on the desired stringency; (i)hybridization solution of 6×SSC and 1% SDS or 3 M TMACl, 0.01 M sodiumphosphate (pH 6.8), 1 mM EDTA (pH 7.6), 0.5% SDS, 100 μg/ml denaturedsalmon sperm DNA and 0.1% nonfat dried milk, hybridization temperatureof 1-1.5° C. below the Tm, final wash solution of 3 M TMACl, 0.01 Msodium phosphate (pH 6.8), 1 mM EDTA (pH 7.6), 0.5% SDS at 1-1.5° C.below the Tm (stringent hybridization conditions) (ii) hybridizationsolution of 6×SSC and 0.1% SDS or 3 M TMACl, 0.01 M sodium phosphate (pH6.8), 1 mM EDTA (pH 7.6), 0.5% SDS, 100 μg/ml denatured salmon sperm DNAand 0.1% nonfat dried milk, hybridization temperature of 2-2.5° C. belowthe Tm, final wash solution of 3 M TMACl, 0.01 M sodium phosphate (pH6.8), 1 mM EDTA (pH 7.6), 0.5% SDS at 1-1.5° C. below the Tm, final washsolution of 6×SSC, and final wash at 22° C. (stringent to moderatehybridization conditions); and (iii) hybridization solution of 6×SSC and1% SDS or 3 M TMACl, 0.01 M sodium phosphate (pH 6.8), 1 mM EDTA (pH7.6), 0.5% SDS, 100 μg/ml denatured salmon sperm DNA and 0.1% nonfatdried milk, hybridization temperature at 2.5-3° C. below the Tm andfinal wash solution of 6×SSC at 22° C. (moderate hybridizationsolution).

Northern Blot analysis: This method involves the detection of aparticular RNA in a mixture of RNAs. An RNA sample is denatured bytreatment with an agent (e.g., formaldehyde) that prevents hydrogenbonding between base pairs, ensuring that all the RNA molecules have anunfolded, linear conformation. The individual RNA molecules are thenseparated according to size by gel electrophoresis and transferred to anitrocellulose or a nylon-based membrane to which the denatured RNAsadhere. The membrane is then exposed to labeled DNA probes. Probes maybe labeled using radio-isotopes or enzyme linked nucleotides. Detectionmay be using autoradiography, colorimetric reaction orchemiluminescence. This method allows both quantitation of an amount ofparticular RNA molecules and determination of its identity by a relativeposition on the membrane which is indicative of a migration distance inthe gel during electrophoresis.

RT-PCR analysis: This method uses PCR amplification of relatively rareRNAs molecules. First, RNA molecules are purified from the cells andconverted into complementary DNA (cDNA) using a reverse transcriptaseenzyme (such as an MMLV-RT) and primers such as, oligo dT, randomhexamers or gene specific primers. Then by applying gene specificprimers and Taq DNA polymerase, a PCR amplification reaction is carriedout in a PCR machine. Those of skills in the art are capable ofselecting the length and sequence of the gene specific primers and thePCR conditions (i.e., annealing temperatures, number of cycles and thelike) which are suitable for detecting specific RNA molecules. It willbe appreciated that a semi-quantitative RT-PCR reaction can be employedby adjusting the number of PCR cycles and comparing the amplificationproduct to known controls.

RNA in situ hybridization stain: In this method DNA or RNA probes areattached to the RNA molecules present in the cells. Generally, the cellsare first fixed to microscopic slides to preserve the cellular structureand to prevent the RNA molecules from being degraded and then aresubjected to hybridization buffer containing the labeled probe. Thehybridization buffer includes reagents such as formamide and salts(e.g., sodium chloride and sodium citrate) which enable specifichybridization of the DNA or RNA probes with their target mRNA moleculesin situ while avoiding non-specific binding of probe. Those of skills inthe art are capable of adjusting the hybridization conditions (i.e.,temperature, concentration of salts and formamide and the like) tospecific probes and types of cells. Following hybridization, any unboundprobe is washed off and the slide is subjected to either a photographicemulsion which reveals signals generated using radio-labeled probes orto a colorimetric reaction which reveals signals generated usingenzyme-linked labeled probes.

In situ RT-PCR stain: This method is described in Nuovo G J, et al.[Intracellular localization of polymerase chain reaction (PCR)-amplifiedhepatitis C cDNA. Am J Surg Pathol. 1993, 17: 683-90] and Komminoth P,et al. [Evaluation of methods for hepatitis C virus detection inarchival liver biopsies. Comparison of histology, immunohistochemistry,in situ hybridization, reverse transcriptase polymerase chain reaction(RT-PCR) and in situ RT-PCR. Pathol Res Pract. 1994, 190: 1017-25].Briefly, the RT-PCR reaction is performed on fixed cells byincorporating labeled nucleotides to the PCR reaction. The reaction iscarried on using a specific in situ RT-PCR apparatus such as thelaser-capture microdissection PixCell I LCM system available fromArcturus Engineering (Mountainview, Calif.).

Oligonucleotide microarray—In this method oligonucleotide probes capableof specifically hybridizing with the polynucleotides of the presentinvention are attached to a solid surface (e.g., a glass wafer). Eacholigonucleotide probe is of approximately 20-25 nucleic acids in length.To detect the expression pattern of the polynucleotides of the presentinvention in a specific cell sample (e.g., blood cells), RNA isextracted from the cell sample using methods known in the art (usinge.g., a TRIZOL solution, Gibco BRL, USA). Hybridization can take placeusing either labeled oligonucleotide probes (e.g., 5′-biotinylatedprobes) or labeled fragments of complementary DNA (cDNA) or RNA (cRNA).Briefly, double stranded cDNA is prepared from the RNA using reversetranscriptase (RT) (e.g., Superscript II RT), DNA ligase and DNApolymerase I, all according to manufacturer's instructions (InvitrogenLife Technologies, Frederick, Md., USA). To prepare labeled cRNA, thedouble stranded cDNA is subjected to an in vitro transcription reactionin the presence of biotinylated nucleotides using e.g., the BioArrayHigh Yield RNA Transcript Labeling Kit (Enzo, Diagnostics, AffymetrixSanta Clara Calif.). For efficient hybridization the labeled cRNA can befragmented by incubating the RNA in 40 mM Tris Acetate (pH 8.1), 100 mMpotassium acetate and 30 mM magnesium acetate for 35 minutes at 94° C.Following hybridization, the microarray is washed and the hybridizationsignal is scanned using a confocal laser fluorescence scanner whichmeasures fluorescence intensity emitted by the labeled cRNA bound to theprobe arrays.

For example, in the Affymetrix microarray (Affymetrix®, Santa Clara,Calif.) each gene on the array is represented by a series of differentoligonucleotide probes, of which, each probe pair consists of a perfectmatch oligonucleotide and a mismatch oligonucleotide. While the perfectmatch probe has a sequence exactly complimentary to the particular gene,thus enabling the measurement of the level of expression of theparticular gene, the mismatch probe differs from the perfect match probeby a single base substitution at the center base position. Thehybridization signal is scanned using the Agilent scanner, and theMicroarray Suite software subtracts the non-specific signal resultingfrom the mismatch probe from the signal resulting from the perfect matchprobe.

Methods of Detecting the Polypeptides on the Protein Level

When the polypeptide is expressed solely on the membrane (e.g. ITGA3)and not in other cell compartments, whole cell extracts may be analyzed.In this scenario lysed cells may be used for the detection of thepolypeptides.

Preferably, when the polypeptide is not expressed solely on the membraneand is expressed in other cell compartments, membrane extracts are usedfor methods which require the generation of cellular extracts. In situmethods, such as immunostaining and FACS may be used regardless whetherthe polypeptide is expressed solely on the membrane or not.

Determining expression of the polypeptides on the protein level istypically effected using an antibody capable of specifically interactingwith same. Methods of detecting the above described proteins includeimmunoassays which include but are not limited to competitive andnon-competitive assay systems using techniques such as Western blots,radioimmunoassays, ELISA (enzyme linked immunosorbent assay), “sandwich”immunoassays, and immunoprecipitation assays and immunohistochemicalassays as detailed herein below.

Below is a list of techniques which may be used to determine the levelof the proteins described herein above on the protein level.

Enzyme linked immunosorbent assay (ELISA): This method involves fixationof a sample (e.g., fixed cells or a proteinaceous solution) containing aprotein substrate to a surface such as a well of a microtiter plate. Asubstrate specific antibody coupled to an enzyme is applied and allowedto bind to the substrate. Presence of the antibody is then detected andquantitated by a colorimetric reaction employing the enzyme coupled tothe antibody. Enzymes commonly employed in this method includehorseradish peroxidase and alkaline phosphatase. If well calibrated andwithin the linear range of response, the amount of substrate present inthe sample is proportional to the amount of color produced. A substratestandard is generally employed to improve quantitative accuracy.

Western blot: This method involves separation of a substrate from otherprotein by means of an acrylamide gel followed by transfer of thesubstrate to a membrane (e.g., nylon or PVDF). Presence of the substrateis then detected by antibodies specific to the substrate, which are inturn detected by antibody binding reagents. Antibody binding reagentsmay be, for example, protein A, or other antibodies. Antibody bindingreagents may be radiolabeled or enzyme linked as described hereinabove.Detection may be by autoradiography, colorimetric reaction orchemiluminescence. This method allows both quantitation of an amount ofsubstrate and determination of its identity by a relative position onthe membrane which is indicative of a migration distance in theacrylamide gel during electrophoresis.

Radio-immunoassay (RIA): In one version, this method involvesprecipitation of the desired protein (i.e., the substrate) with aspecific antibody and radiolabeled antibody binding protein (e.g.,protein A labeled with I¹²⁵) immobilized on a precipitable carrier suchas agarose beads. The number of counts in the precipitated pellet isproportional to the amount of substrate.

In an alternate version of the RIA, a labeled substrate and an unlabeledantibody binding protein are employed. A sample containing an unknownamount of substrate is added in varying amounts. The decrease inprecipitated counts from the labeled substrate is proportional to theamount of substrate in the added sample.

Fluorescence activated cell sorting (FACS): This method involvesdetection of a substrate in situ in cells by substrate specificantibodies. The substrate specific antibodies are linked tofluorophores. Detection is by means of a cell sorting machine whichreads the wavelength of light emitted from each cell as it passesthrough a light beam. This method may employ two or more antibodiessimultaneously. It will be appreciated that when the protein is notexpressed selectively on the cell membrane, care should be taken toavoid the antibody penetrating the cell membrane. Immunohistochemicalanalysis: This method involves detection of a substrate in situ in fixedcells by substrate specific antibodies. The substrate specificantibodies may be enzyme linked or linked to fluorophores. Detection isby microscopy and subjective or automatic evaluation. If enzyme linkedantibodies are employed, a colorimetric reaction may be required. Itwill be appreciated that immunohistochemistry is often followed bycounterstaining of the cell nuclei using for example Hematoxyline orGiemsa stain. It will be appreciated that when the protein is notexpressed selectively on the cell membrane, care should be taken toavoid the antibody penetrating the cell membrane.

In situ activity assay: According to this method, a chromogenicsubstrate is applied on the cells containing an active enzyme and theenzyme catalyzes a reaction in which the substrate is decomposed toproduce a chromogenic product visible by a light or a fluorescentmicroscope.

As mentioned, the identifying/diagnosing/staging is carried out byanalyzing an amount or activity of the polypeptides in a cell sample ofthe subject, wherein a difference in an amount or activity thereofbeyond a predetermined threshold with respect to a control cell sampleis indicative of the disease. It will be appreciated that the amount ofchange may correspond with a degree or a stage of the disease. Thus,larger differences may indicate a later stage of the disease with apoorer prognosis, whereas lower differences may indicate an early stageof the disease with a better prognosis.

The patient sample typically comprises cells. It may be part of a tissuesample, retrieved during a biopsy. Alternatively, the sample may be abodily fluid, e.g. blood, urine, saliva, CSF, plasma etc.

For diagnosis of cancer, the cell sample may comprise cells of theprimary tumor and/or metastatic effusion thereof.

The predetermined level may be established based on results from control(non-diseased) cells.

The control cell sample typically depends on the patient sample beinganalyzed. Thus, for example, in the case of colon cancer, the controlsample may comprise colon cells of a healthy individual (or at least onenot suffering from colon cancer) or from a known stage of colon cancer(e.g. non-metastatic stage). In the case of breast cancer, the controlsample may comprise breast cells of a healthy individual (or at leastone not suffering from breast cancer) or from a known stage of breastcancer.

The control cells are typically normally differentiated, non-senescentcells, preferably of the same tissue and specimen as the tested cells.Typically, the amount of change in expression of the polypeptides isstatistically significant.

Preferably, the difference is at least 10%, 20%, 30%, 40%, 50%, 80%,100% (i.e., two-fold), 3 fold, 5 fold or 10 fold different as comparedto the control cells.

It will be appreciated that the control data may also be taken fromdatabases and literature.

On obtaining the results of the analysis, the subject is typicallyinformed. Additional diagnostic tests may also be performed so as tocorroborate the results of the diagnosing (e.g. gold standard tests,assessing the aggressiveness of the tumor, the patient's health andsusceptibility to treatment, etc.).

Imaging studies such as CT and/or MRI may be obtained to furtherdiagnose the disease.

In addition, when the disease is cancer, the diagnosis or choice oftherapy may be determined by further assessing the size of the tumor, orthe lymph node stage or both, optionally together or in combination withother risk factors.

The present inventors propose that based on the results of thediagnosis, a suitable therapy may be selected—i.e. personalizedmedicine.

As mentioned, the present inventors showed that down-regulation ofcell-surface Grp94 decreases NK-cell mediated cytotoxicity towardsenescent cells and decreased susceptibility of senescent cells forelimination by monocytes.

Thus, the present inventors propose that polypeptides which areexpressed on the surface of senescent cells may be used to elicit orboost an immune response to a senescent cell.

Thus, according to another aspect of the present invention there isprovided a method of eliciting or boosting an immune response to asenescent cell in a subject comprising administering to the subject apharmaceutical composition comprising at least one polypeptide or apolynucleotide encoding same selected from the group consisting ofHSP90B1, DBN1, PRKCSH, SPTBN1, NPM1 and a polypeptide set forth in Table1, wherein the pharmaceutical composition does not comprise senescentcells, thereby eliciting or boosting the immune response to thesenescent cell.

The immune response may comprise clearance of the senescent cell by theimmune response (e.g. a helper T cell or a cytotoxic T-cell response).

The present inventors contemplate administering at least one, two,three, four, five, six, seven, eight, nine, ten or more of thepolypeptides of this aspect of the present invention.

The polypeptides may be provided as full length polypeptides or asantigenic fragments thereof.

As used herein, the term “antigenic fragment” refers to an immunogenicportion of the full-length polypeptide. Such antigenic fragments maycomprise at least 5, 10, 20, 25, 30, 35, 40, 45, 50, 55, or 60 or morecontiguous amino acids (or any number of contiguous amino acids between5-60, including 5-10, 10-15, 15-20, 20-25, 25-30, 30-35, 35-40, 40-45,45-50, 40-55, or 55-60, or more than 60 contiguous amino acids) of thepolypeptides. In a more particular embodiment, antigenic fragmentcomprises at least 20 contiguous amino acids. An antigenic fragment of amature or full-length polypeptide has one or more epitopes that induce aspecific immune response, which may comprise production of antibodiesthat specifically bind to the antigenic fragment and to the immunogenicportion within the mature and full-length polypeptide from which theantigenic peptide is derived, and to a senescent cell that expresses thepolypeptide.

The polypeptides or fragments thereof of the present invention may beadministered per se to induce an immune response, or alternatively, aspart of a composition i.e. vaccine, which comprises an immunologicallyacceptable carrier.

It will be appreciated that the polypeptides may be administered in theform of an expression construct which comprises the correspondingnucleic acid sequence to the polypeptide. The expression construct maybe administered instead of the polypeptides themselves (e.g. in a primeboost protocol) or in addition to the polypeptides of the presentinvention. It will be further appreciated that the polypeptides may beexpressed in a cell population (e.g. dendritic cells) and the cellpopulation may be provided. According to a particular embodiment, thepolypeptides are not administered as part of a senescent cell populationor membrane fraction thereof.

Suitable agents that provide a target antigen include recombinantvectors, for example, bacteria, viruses, and naked DNA. Recombinantvectors are prepared using standard techniques known in the art, andcontain suitable control elements operably linked to the nucleotidesequence encoding the target antigen. See, for example, Plotkin, et al.(eds.) (2003) Vaccines, 4.sup.th ed., W.B. Saunders, Co., Phila., Pa.;Sikora, et al. (eds.) (1996) Tumor Immunology Cambridge UniversityPress, Cambridge, UK; Hackett and Ham (eds.) Vaccine Adjuvants, HumanaPress, Totowa, N.J.; Isaacson (eds.) (1992) Recombinant DNA Vaccines,Marcel Dekker, NY, N.Y.; Morse, et al. (eds.) (2004) Handbook of CancerVaccines, Humana Press, Totowa, N.J.), Liao, et al. (2005) Cancer Res,65:9089-9098; Dean (2005) Expert Opin. Drug Deliv. 2:227-236; Arlen, etal. (2003) Expert Rev. Vaccines 2:483-493; Dela Cruz, et al. (2003)Vaccine 21:1317-1326; Johansen, et al. (2000) Eur. J. Pharm. Biopharm.50:413-417; Excler (1998) Vaccine 16:1439-1443; Disis, et al. (1996) J.Immunol. 156:3151-3158). Peptide vaccines are described (see, e.g.,McCabe, et al. (1995) Cancer Res. 55:1741-1747; Minev, et al. (1994)Cancer Res. 54:4155-4161; Snyder, et al. (2004) J. Virology78:7052-7060. Virus-derived vectors include viruses, modified viruses,and viral particles (see, e.g., U.S. Pat. No. 8,926,993, incorporatedherein by reference). The virus-derived vectors can be administereddirectly to a mammalian subject, or can be introduced ex vivo into anantigen presenting cell (APC), where the APC is then administered to thesubject.

Viral vectors may be based on, e.g., Togaviruses, including alphavirusesand flaviviruses; alphaviruses, such as Sindbis virus, Sindbis strainSAAR86, Semliki Forest virus (SFV), Venezuelan equine encephalitis(VEE), Eastern equine encephalitis (EEE), Western equine encephalitis,Ross River virus, Sagiyami virus, O'Nyong-nyong virus, Highlands Jvirus. Flaviviruses, such as Yellow fever virus, Yellow fever strain17D, Japanese encephalitis, St. Louis encephalitis, Tick-borneencephalitis, Dengue virus, West Nile virus, Kunjin virus (subtype ofWest Nile virus); arterivirus such as equine arteritis virus; andrubivirus such as rubella virus, herpesvirus, modified vaccinia Ankara(MVA); avipox viral vector; fowlpox vector; vaccinia virus vector;influenza virus vector; adenoviral vector, human papilloma virus vector;bovine papilloma virus vector, and so on. Viral vectors may be based onan orthopoxvirus such as variola virus (smallpox), vaccinia virus(vaccine for smallpox), Ankara (MVA), or Copenhagen strain, camelpox,monkeypox, or cowpox. Viral vectors may be based on an avipoxvirusvirus, such as fowlpox virus or canarypox virus.

Adenoviral vectors and adeno-associated virus vectors (AAV) areavailable, where adenoviral vectors include adenovirus serotype 5(adeno5; Ads), adeno6, adeno11, and adeno35. Available are at least 51human adenovirus serotypes, classified into six subgroups (subgroups A,B, C, D, E, and F). Adenovirus proteins useful, for example, inassessing immune response to an “empty” advenoviral vector, includehexon protein, such as hexon 3 protein, fiber protein, and penton baseproteins, and human immune responses to adenoviral proteins have beendescribed (see, e.g., Wu, et al. (2002) J. Virol. 76:12775-12782,Mascola (2006) Nature 441:161-162; Roberts, et al. (2006) Nature441:239-243).

General methods to prepare immunogenic or vaccine compositions aredescribed in Remington's Pharmaceutical Science; Mack Publishing CompanyEaston, Pa. (latest edition). To increase immunogenicity, thepolypeptides of the present invention may be adsorbed to or conjugatedto beads such as latex or gold beads, ISCOMs, and the like. Immunogeniccompositions may comprise adjuvants, which are substance that can beadded to an immunogen or to a vaccine formulation to enhance theimmune-stimulating properties of the immunogenic moiety. Liposomes arealso considered to be adjuvants (Gregoriades, G. et al., ImmunologicalAdjuvants and Vaccines, Plenum Press, New York, 1989) Examples ofadjuvants or agents that may add to the effectiveness of proteinaceousimmunogens include aluminum hydroxide, aluminum phosphate, aluminumpotassium sulfate (alum), beryllium sulfate, silica, kaolin, carbon,water-in-oil emulsions, and oil-in-water emulsions. A preferred type ofadjuvant is muramyl dipeptide (MDP) and various MDP derivatives andformulations, e.g.,N-acetyl-D-glucosaminyl-(.beta.1-4)-N-acetylmuramyl-L-alanyl-D-isoglutamine(GMDP) (Hornung, R L et al. Ther Immunol 1995 2:7-14) or ISAF-1 (5%squalene, 2.5% pluronic L121, 0.2% Tween 80 in phosphate-bufferedsolution with 0.4 mg of threonyl-muramyl dipeptide; see Kwak, L W et al.(1992) N. Engl. J. Med., 327:1209-1238). Other useful adjuvants are, orare based on, cholera toxin, bacterial endotoxin, lipid X, wholeorganisms or subcellular fractions of the bacteria Propionobacteriumacnes or Bordetella pertussis, polyribonucleotides, sodium alginate,lanolin, lysolecithin, vitamin A, saponin and saponin derivatives suchas QS21 (White, A. C. et al. (1991) Adv. Exp. Med. Biol., 303:207-210)which is now in use in the clinic (Helling, F et al. (1995) Cancer Res.,55:2783-2788; Davis, T A et al. (1997) Blood, 90: 509), levamisole,DEAE-dextran, blocked copolymers or other synthetic adjuvants. A numberof adjuvants are available commercially from various sources, forexample, Merck Adjuvant 65 (Merck and Company, Inc., Rahway, N.J.) orFreund's Incomplete Adjuvant and Complete Adjuvant (Difco Laboratories,Detroit, Mich.), Amphigen (oil-in-water), Alhydrogel (aluminumhydroxide), or a mixture of Amphigen and Alhydrogel. Aluminum isapproved for human use.

The present invention also contemplates antiserum induced in one subjectusing the polypeptides or fragments thereof of the present invention,removed from that subject and used to treat another subject by passiveimmunization or transfer of the antibodies. For disclosure of suchpassive immunization with patient sera, neutralizing antisera or mAbs,see Nishimura Y et al. (2003) Proc Natl Acad Sci USA 100:15131-36;Mascola J R (2003) Curr Mol Med. 3:209-16; Ferrantelli F et al. (2003)AIDS 17:301-9; Ferrantelli F et al (2002) Curr Opin Immunol. 14:495-502;Xu W et al. (2002) Vaccine 20:1956-60; Nichols C N et al. (2002) AIDSRes Hum Retrovir. 8:49-56; Cho M W et al. (2000) J. Virol. 74:9749-54;Mascola R et al. (2000) Nat Med. 6:207-10; Andrus. L et al. (1998) J.Inf. Dis. 77: 889-897; Parren P W (1995) AIDS 9:F1-6; Hinkula J et al.(1994) J Acquir Immune Defic Syndr. 7:940-51; Prince A M et al. (1991)AIDS Res Hum Retrovir 7:971-73; Emini E A et al. (1990) J. Virol.64:3674-84, all incorporated by reference.

The amount of polypeptide or fragment to be administered to induce animmune response depends on the precise polypeptide selected, the healthand weight of the recipient, the route of administration, the existenceof other concurrent treatment, if any, the frequency of treatment, thenature of the effect desired, and the judgment of the skilledpractitioner.

An exemplary dose for treating a subject is an amount of up to about 100milligrams of active polypeptide per kilogram of body weight. A typicalsingle dosage of the polypeptide or chimeric protein is between about 1ng and about 100 mg/kg body weight, and preferably from about 10 μg toabout 50 mg/kg body weight. A total daily dosage in the range of about0.1 milligrams to about 7 grams is preferred for intravenousadministration. A useful dose of an antibody for passive immunization isbetween 10-100 mg/kg. It has been suggested (see references cited abovefor passive immunity) that an effective in vivo dose of anantibody/antiserum is between about 10- and 100-fold more than aneffective neutralizing concentration or dose in vitro. These dosages canbe determined empirically in conjunction with the present disclosure andstate-of-the-art. The polypeptides of the present invention may beadministered alone or in conjunction with other therapeutics directed tothe treatment of the disease or condition.

The subject vaccines find use in methods for eliciting or boosting acellular immune response, e.g., a helper T cell or a cytotoxic T-cellresponse to senescent cells. The vaccine of the present invention may beused, for example, both for immunization and to boost immunity afterexposure. As such, the subject vaccines find use as both prophylacticand therapeutic vaccines to induce immune responses that are specificfor senescent cells that are relevant to various disease conditions.

The vaccine may contain other antigenic polypeptides, e.g. comprised ina tumor cell lysate, an irradiated tumor cell, an antigen-presentingcell pulsed with peptides of the target antigen (e.g. a dendritic cell).

The vaccine of this aspect of the present invention may also comprise anagent which enhances the immunogenicity of the immunogen e.g. a helperantigen or carrier moiety. A helper antigen includes a T cell helperantigen, which is an antigen that is recognized by a T helper cell andevokes an immune response in a T helper cell. T helper cells arelymphocytes that are involved in activating and directing other immunecells such as cytotoxic T cells, B cells, and/or macrophages. Carriermoieties have been long known in the immunology art and include withoutlimitation, keyhole limpet hemocyanin, bovine serum albumin, cationizedBSA, or ovalbumin. For human use, toxoids of bacterial proteins (e.g.,tetanus toxoid, diphtheria toxoid, cholera toxoid, and the like) aretypically employed as carrier proteins.

In certain embodiments, the immunogen comprises at least one senescentcell associated antigen or at least one antigenic fragment thereof and ahelper antigen or carrier moiety that is linked, conjugated, or attachedto the antigen or antigenic fragment thereof. The helper antigen orcarrier moiety may be recombinantly expressed in frame and directlylinked to a senescent cell associated antigen or fragment thereof. Incertain embodiments, a fusion protein comprising at least two senescentcell associated antigens or at least two antigenic fragments thereof ora combination of same may also comprise a helper antigen or carriermoiety. Alternatively, the helper antigen or carrier moiety may bechemically conjugated, linked, or attached to the senescent cellassociated antigen or fragment thereof. In still another embodiment, thehelper antigen or carrier moiety may be formulated together with anyimmunogen described herein but not covalently or non-covalently bound tothe immunogen to form an immunogenic composition.

In another embodiment, the immunogenic compositions described hereininclude a co-stimulatory polypeptide. in certain embodiments, theimmunogen comprises at least one senescent cell associated antigen (asdescribed herein) or at least one antigenic fragment thereof and aco-stimulatory molecule that is linked, conjugated, or attached to theantigen or antigenic fragment thereof. The co-stimulatory molecule maybe recombinantly expressed in frame and directly linked to a senescentcell associated antigen or fragment thereof. In certain embodiments, afusion protein comprising at least two senescent cell associated antigenor at least two antigenic fragments thereof or a combination of same mayalso comprise a co-stimulatory molecule. Alternatively, theco-stimulatory molecule may be chemically conjugated, linked, orattached to the senescent cell associated antigen or fragment thereof.In still another embodiment, the co-stimulatory molecule may beformulated together with any immunogen described herein but notcovalently or non-covalently bound to the immunogen to form theimmunogenic composition.

Exemplary co-stimulatory molecules include, by way of example, GM-CSF,IL-2, IL-4, IL-6, IL-7, IL-15, IL-21, IL-23, TNFa, B7.1 (CD80), B7.2(CD86), 41BB, CD40 ligand (CD40L), drug-inducible CD40 (iCD40), and thelike. When an immunogenic composition comprises a polynucleotideencoding the co-stimulatory molecule, or a recombinant expression viruscomprising the polynucleotide, expression of the co-stimulatory moleculeis typically under the control of one or more regulatory elementsselected to direct the expression of the coding sequences in a cell ofchoice, such as a dendritic cell.

Recombinantly engineered antigen-presenting cells such as dendriticcells, for example, may be modified by recombinant technology to expressincreased levels of antigen presenting machinery, adhesion and/orco-stimulatory molecules, including MHC class I/antigen complexes, MHCclass II/antigen complexes, CD1, hsp70-90, CD9, CD63, CD81, CD11b,CD11c, CD40, CD54 (ICAM-1), CD63, CD80, CD86, 41BBL, OX40L, chemokinereceptor CCR1-10 and CXCR1-6, mannose-rich C-type lectin receptor DEC205and Toll-like receptors TLR4 and TLR9 or membrane-bound TGF-β. Theexosomes derived from these recombinantly engineered antigen presentingcells will express these additional molecules and can transfer them tothe T helper cells, T regulatory cells, or dendritic cells uponabsorption.

As mentioned, the present invention contemplates antigen-presentingcells (APCs), e.g., dendritic cells (DCs), that include senescentcell-associated antigens, as described herein above, for example, bybeing presented on the surface of the antigen-presenting cells.

Dendritic cells play a critical role in coordinating innate and adaptiveimmune responses. DCs are bone-marrow derived cells characterized bydendritic morphology and high mobility that are seeded in all tissues.DCs are specialized antigen presenting cells that are capable ofcapturing and processing antigens, migrating from the periphery to alymphoid organ, and presenting the antigens in a MHC-restricted mannerto naive T-cells (see, e.g., Banchereau & Steinman, 1998, Nature392:245-252; Steinman et al., 2003, Ann. Rev. Immunol, 21:685-711).Immature DCs are capable of processing and presenting antigens, whichleads to immune regulation and/or suppression. Maturation (activation)of DCs is required to induce differentiation of antigen-specific T cellsinto effector T cells (see, Palucka et al, 2012, Nat. Rev. Cancer12:265-277). Mature DCs express high levels of MHC-antigen complex andother co-stimulatory molecules, such as CD40, B7-1, B7-2, and CD 1a(see, e.g., Steinman, 1991, Ann. Rev. Immunol. 9:271-296; Banchereau &Steinman, 1998, Nature 392:245-252). These molecules play key roles instimulating T cells. Due to their properties, DC-based vaccinationstrategies have been developed in cancer (see, e.g., Heiser et al, 2001,Cancer Res. 61:338; Heiser et al, 2001, J. Immunol. 166:2953; Milazzo etal, 2002, Blood 101:977; Zu et al, 2003, Cancer Res. 63:2127). Likewise,DC based immunogens (vaccines) may be able to elicit CD8⁺ T cellscapable of recognizing peptide-MHC Class complexes on senescent cellsand target them for destruction.

Dendritic cells may be obtained from various sources using methods knownin the art. DC precursors may be purified from peripheral blood (see,e.g., Fong et al., 2003, Annu Rev. Immunol. 15: 138). DCs may be also bedifferentiated from peripheral blood monocytes or CD34⁺ hematopoieticprogenitor cells ex vivo (see, e.g., Sallusto et al, 1994, J. Exp. Med.179: 1109; Banchereau et al, 2001, Cancer Res. 61:6451; Makensen et al,2000, Int. J. Cancer 86:385). Methods for in vitro proliferation ofdendritic cells from DC precursors and their use as immunogens aredescribed in U.S. Pat. Nos. 5,851,756; 5,994,126; 6,475,483; and8,283,163 each of which is incorporated herein by reference in itsentirety. A method for isolating DCs from human peripheral blood isdescribed in U.S. Pat. No. 5,643,786, incorporated herein by referencein its entirety. U.S. Patent Publication 2006/0063255, U.S. PatentPublication 2006/0057129, and U.S. Pat. No. 7,247,480, each of which isincorporated herein by reference in its entirety, describe methods formaking dendritic cell vaccines from human embryonic stem cells.

Methods of isolating APCs, such as dendritic cells, are known in theart. Procedures such as repetitive density gradient separation,fluorescence activated cell sorting techniques, positive selection,negative selection, or a combination thereof are routinely used toobtain enriched populations of DCs. Methods for isolating DCs may befound in O'Doherty et al, 1993, J. Exp. Med. 178: 1067-78; Young andSteinman, 1990, J. Exp. Med. 171:1315-32; Freudenthal et al, 1990, Proc.Natl. Acad. Sci. USA 57:7698-7702; Markowicz and Engleman, 1990, J.Clin. Invest. 85:955-961; Mehta-Damani et al, 1994, J. Immunol.153:996-1003; Thomas et al, 1993, J. Immunol. 151:6840-6852.

Dendritic cells may be loaded with specific antigens ex vivo and thenadministered to a subject (see, e.g., Banchereau et al, 2005, Nat. Rev.Immunol. 5:296-306; Figdor et al, 2004, Nat. Med. 10:475-480, each ofwhich is incorporated herein by reference in its entirety). Variousmethods for loading antigens to DCs have been described and are known inthe art. R A encoding a specific antigen may be pulsed into dendriticcells before administration to a subject by electroporation, cationiclipids, cationic peptides or using dendrimers (see, e.g., Boczkwoski etal. 1996, J. Exp. Med. 184:465; Heiser et al, 2001, Cancer Res. 61:338;Heiser et al, J. Immunol. 2001, 166:2953; U.S. Patent Publication2006/0063255; Choi et al, 2005, Cell Cycle 4:669). DCs may also beloaded with protein or peptide that is purified or isolated from atarget cell, chemically synthesized, or recombinantly expressed. Nucleicacid vectors encoding a specific antigen may also be used for DC loading(see, e.g., Frolkis et al., 2003, Cancer Gene Ther. 10:239). Exemplaryvectors include plasmids, cationic lipid complexes, viral vectors, cDNAencoding antigen loaded onto dendrimers, or other small particulatesthat enhance uptake by phagocytic cells. U.S. Pat. Nos. 6,300,090 and6,455,299 describe using non-replicating viral vectors comprisingsequence encoding an antigen for infecting dendritic cells, resulting inantigen presentation on the DC surface.

Alternatively, DCs may be loaded with specific antigens in vivo.

Antigens may be delivered directly to DCs using chimeric proteins thatare comprised of a DC receptor-specific antibody fused to a selectedantigen (see, e.g., Bonifaz et al, 2004, J. Exp. Med. 199:815-824;Bonifaz et al, 2004, J. Exp. Med. 196: 1627-1638; Hawiger et al, 2001,J. Exp. Med. 194:769-780; each of which is incorporated herein byreference in its entirety). U.S. Patent Publication 2012/0070462,incorporated herein by reference in its entirety, describes targetedantigen delivery to dendritic cells using recombinant viral vectorscomprising a polynucleotide encoding the antigen and a targetingmolecule, which binds to a DC-specific surface marker (e.g., DC-SIGN).

Antigenic peptides useful for presentation by DCs for vaccination arepeptides that stimulate a T cell mediated immune response (e.g.,cytotoxic T cell response) by presentation to T cells on MHC molecules.Useful antigenic peptides and proteins for use in the present disclosureinclude those derived from senescent cells (e.g., senescentcell-associated antigens). Depending on the method of DC loadingutilized, a senescent cell-associated antigen may be presented in avariety of forms. In some embodiments, a senescent cell-associatedantigen is presented as a senescent cell lysate to DCs. In otherembodiments, senescent cell-associated antigens are obtained by acidelution of peptides presented on MHC molecules of the senescent cellsurface. For example, senescent cells are washed with an isotonicsolution to remove media components. The cells are then treated withacid to dissociate peptides from surface MHCs, and the cells are removedfrom the solution containing the soluble peptides. Antigenic peptidesmay be obtained by chemical synthesis or produced using recombinantmethods with host cells and vector expression systems. A senescent cellassociated antigen may also be delivered as a polynucleotides (RNA orDNA) to a DC directly or indirectly (e.g., via a plasmid or viralvector). The antigenic peptides presented on MHC molecules are typicallyshort peptides and may be 5, 6, 7, 8, 9, or 10 amino acids, for example.

A senescent cell associated antigen introduced into DCs may also bedesigned as a fusion peptide, wherein the antigen is joined to a proteinor peptide sequence that enhances transport into endosomal and otherintracellular compartments involved in Class II histocompatibilityloading. For example, the N-terminus of such a fusion protein maycomprise a suitable heterologous leader or signal sequence for theendosomal compartment and the C-terminus may comprise a transmembraneand luminal component of a member of the LAMP family for lysosomaltargeting (see, e.g., U.S. Pat. No. 5,633,234; WO 02/080851; Sawada etal, 1993, J. Biol. Chem. 268:9014; each of which is incorporated byreference herein in its entirety). Endosomal and lysosomal sortingsignals include tyrosine based signals, dileucine-based signals, acidicclusters, and transmembrane proteins labeled with ubiquitin (see, e.g.,Bonifacino et al, 2003, Annu. Rev. Biochem. 72:395; U.S. Pat. No.6,248,565).

It is expected that during the life of a patent maturing from thisapplication many relevant pharmaceutical agents will be developed andthe scope of the term pharmaceutical agent is intended to include allsuch new technologies a priori.

As used herein the term “about” refers to ±10%.

The terms “comprises”, “comprising”, “includes”, “including”, “having”and their conjugates mean “including but not limited to”.

The term “consisting of” means “including and limited to”.

The term “consisting essentially of” means that the composition, methodor structure may include additional ingredients, steps and/or parts, butonly if the additional ingredients, steps and/or parts do not materiallyalter the basic and novel characteristics of the claimed composition,method or structure.

As used herein, the singular form “a”, “an” and “the” include pluralreferences unless the context clearly dictates otherwise. For example,the term “a compound” or “at least one compound” may include a pluralityof compounds, including mixtures thereof

Whenever a numerical range is indicated herein, it is meant to includeany cited numeral (fractional or integral) within the indicated range.The phrases “ranging/ranges between” a first indicate number and asecond indicate number and “ranging/ranges from” a first indicate number“to” a second indicate number are used herein interchangeably and aremeant to include the first and second indicated numbers and all thefractional and integral numerals therebetween.

As used herein the term “method” refers to manners, means, techniquesand procedures for accomplishing a given task including, but not limitedto, those manners, means, techniques and procedures either known to, orreadily developed from known manners, means, techniques and proceduresby practitioners of the chemical, pharmacological, biological,biochemical and medical arts.

As used herein, the term “treating” includes abrogating, substantiallyinhibiting, slowing or reversing the progression of a condition,substantially ameliorating clinical or aesthetical symptoms of acondition or substantially preventing the appearance of clinical oraesthetical symptoms of a condition.

It is appreciated that certain features of the invention, which are, forclarity, described in the context of separate embodiments, may also beprovided in combination in a single embodiment. Conversely, variousfeatures of the invention, which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable subcombination or as suitable in any other describedembodiment of the invention. Certain features described in the contextof various embodiments are not to be considered essential features ofthose embodiments, unless the embodiment is inoperative without thoseelements.

Various embodiments and aspects of the present invention as delineatedhereinabove and as claimed in the claims section below find experimentalsupport in the following examples.

EXAMPLES

Reference is now made to the following examples, which together with theabove descriptions illustrate some embodiments of the invention in a nonlimiting fashion.

Generally, the nomenclature used herein and the laboratory proceduresutilized in the present invention include molecular, biochemical,microbiological and recombinant DNA techniques. Such techniques arethoroughly explained in the literature. See, for example, “MolecularCloning: A laboratory Manual” Sambrook et al., (1989); “CurrentProtocols in Molecular Biology” Volumes I-III Ausubel, R. M., ed.(1994); Ausubel et al., “Current Protocols in Molecular Biology”, JohnWiley and Sons, Baltimore, Md. (1989); Perbal, “A Practical Guide toMolecular Cloning”, John Wiley & Sons, New York (1988); Watson et al.,“Recombinant DNA”, Scientific American Books, New York; Birren et al.(eds) “Genome Analysis: A Laboratory Manual Series”, Vols, 1-4, ColdSpring Harbor Laboratory Press, New York (1998); methodologies as setforth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and5,272,057; “Cell Biology: A Laboratory Handbook”, Volumes I-III Cellis,J. E., ed. (1994); “Culture of Animal Cells—A Manual of Basic Technique”by Freshney, Wiley-Liss, N. Y. (1994), Third Edition; “Current Protocolsin Immunology” Volumes I-III Coligan J. E., ed. (1994); Stites et al.(eds), “Basic and Clinical Immunology” (8th Edition), Appleton & Lange,Norwalk, Conn. (1994); Mishell and Shiigi (eds), “Selected Methods inCellular Immunology”, W. H. Freeman and Co., New York (1980); availableimmunoassays are extensively described in the patent and scientificliterature, see, for example, U.S. Pat. Nos. 3,791,932; 3,839,153;3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654;3,935,074; 3,984,533; 3,996,345; 4,034,074; 4,098,876; 4,879,219;5,011,771 and 5,281,521; “Oligonucleotide Synthesis” Gait, M. J., ed.(1984); “Nucleic Acid Hybridization” Hames, B. D., and Higgins S. J.,eds. (1985); “Transcription and Translation” Hames, B. D., and HigginsS. J., eds. (1984); “Animal Cell Culture” Freshney, R. I., ed. (1986);“Immobilized Cells and Enzymes” IRL Press, (1986); “A Practical Guide toMolecular Cloning” Perbal, B., (1984) and “Methods in Enzymology” Vol.1-317, Academic Press; “PCR Protocols: A Guide To Methods AndApplications”, Academic Press, San Diego, Calif. (1990); Marshak et al.,“Strategies for Protein Purification and Characterization—A LaboratoryCourse Manual” CSHL Press (1996); all of which are incorporated byreference as if fully set forth herein. Other general references areprovided throughout this document. The procedures therein are believedto be well known in the art and are provided for the convenience of thereader. All the information contained therein is incorporated herein byreference.

Materials and Methods

Cell culture: Human IMR-90 fibroblasts were purchased from ATCC, andMouse embryonic fibroblasts (MEFs) were derived according to standardprocedures (Manipulating the Mouse Embryo: A Laboratory Manual, 3rd ed.A. Nagy, et al., Cold Spring Harbor Laboratory Press, 2003). Cells werecultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with2 mM L-glutamine, 100 units/ml of penicillin, 100 μg/ml of streptomycin,and 10% FBS, in a low oxygen incubator. DIS was induced by etoposidetreatment (E1383, Sigma) at a concentration of 100 μM for 48 hours asdescribed previously (Krizhanovsky et al. Cell 134.4 (2008): 657-667).OIS was induced by infection of IMR-90 cells with pWZL plasmidcontaining oncogenic Hras^(V12).

Identification of cell surface proteins: Cell-surface proteins werepurified from IMR-90 cells, 9 days post insult using commercial kit(Pierce). Briefly, cells were exposed to Sulfo-NHS—SS-biotin, lysed andloaded on columns to capture membrane proteome. Following severalwashing steps, purified cell surface proteins were finally eluted.Samples were then analyzed by Label-free mass spectrometry at theproteomic unit at the Grand Israel National Center for PersonalizedMedicine. Integrative analysis was done by Ingenuity Pathway Analysissoftware (Qiagen). Cell-surface proteins were analyzed according totheir original subcellular localization using the cellular componentbranch of GO by online DAVID v6.7 software.

Validation of MS results: Grp94 translocation to cell surface was firstvalidated by purification of plasma membrane proteome (Qproteome PlasmaMembrane Protein Kit, Qiagen). Equal amounts of cell-surface proteins ortotal cell lysates from growing IMR-90 and senescent IMR-90 wereseparated on 12% SDS-polyacrylamide gels and transferred onto PVDFmembrane (IPVH00010, Millipore). After blocking with 5% BSA in TBST (TBSwith 0.01% Tween 20) for 1 hr, the membranes were probed with antibodiesagainst Grp94 (PA5-24824, Thermo Fisher Scientific), HLA-A,B,C(BioLegend), GAPDH and β-tubulin (both, Santa Cruz) overnight at 4° C.Antibodies were visualized with HRP-based chemiluminescence detectionkit (34080, Thermo Fisher Scientific).

Immunofluorescence assay: Cells were incubated with anti-Grp94 antibody(diluted in DMEM) for 3 h in 37° C., washed 3 times with warm PBS andincubated with goat anti-rabbit Alexa647 conjugated antibody(111-605-003, Jackson immuno-research) for 30 minutes, washed 3 timeswith warm PBS and visualized in Olympus IX81 microscope and XM10 cameraand processed using ImageJ v1.47 software.

Flow-Cytometry Assay

IMR-90 cells were gently dissociated from plates using TripLE expressreagent (12604-013, Thermo Fisher Scientific), and maintained in coldFACS buffer (PBS containing 1% FCS and 0.1% Sodium Azid) throughout allprocedure. Cells were incubated with anti-Grp94 antibody (1:20 in FACSbuffer), followed by goat anti-rabbit Alexa647 conjugated antibody. DAPIwas shortly introduced in order to exclude dead cells. Cells wereanalyzed in a SORP-LSRII instrument (BD Biosciences). Data was collectedfrom at least 20,000 single-cell events. Cells were gated by their size(FSC/SSC), DAPI negative (live cells) and then analyzed for theirfluorescence intensity at the wavelength of 647 (cell-surface Grp94level), using FlowJo v10 software.

GPM1 inhibitor: GPM1 was synthesized by the Organic synthesis unit atthe Weizmann institute as was described previously (Kim et al. 2012).Final product was characterized by NMR. GPM1 were dissolved in 5% DMSOin phosphate-buffered saline (PBS) and control vehicle was 5% DMSO inPBS.

Cytotoxicity assays: For in-vitro cytotoxicity assays target cells wereplated in 12-well plates at 4×10⁴ cells per well; 1×10⁵ NK-92 cells weresubsequently added to each well. Following 2 h of co-incubation, NK-92cells were washed gently and the cytotoxicity was determined based onthe viability of remaining adherent cells. To block Grp94-mediatedcytotoxicity, ON-TARGETplus SMARTpool small-interfering RNA targetingHSP90B1 and the non-targeting (control) pool were transfected intosenescent IMR-90 cells with Dharmafect 1 reagent (all from Dharmacon,Lafayette, Colo., USA). Transfections were performed overnight, and 4days later cytotoxicity assays were executed. Viability was determinedby using PrestoBlue reagent kit (A13262, Life Technologies Ltd.), andwas calculated relatively to control DMSO treated cells.

For phagocytosis assay Etopoide-treated IMR-90 cells were plated in12-well plates at 4×10⁴ cells per well. Cells were incubated 24 hrs withGPM1 at the concentration of 0, 10 or 100 μg/ml, and then co-cultureswith MM6 cells for 6 days. Assessment of remaining cells was determinedby crystal violet staining.

Statistical analysis: Statistical analysis of the results was analyzedusing one-sided two-sample t-tests or one-way ANOVA followed by Tukeypost-hoc test. Data are expressed as means±S.E.M, a P value<0.05 wasconsidered significant.

Results

Senescent cells are known to express several common ligands which arerequired for natural killer immune cells recognition and subsequentcytotoxicity [5]. In order to identify the fraction proteome which isbeing expressed on cell surface of senescent cells, the presentinventors analyzed membrane proteins using high-throughput proteomicsanalysis. Normal human fibroblasts cells IMR90 were used to execute twodistinct sets of experiments; exposure to the chemotherapeutic agentEtoposide for 48 hrs, which results in DNA Damage Induced Senescence(DIS), and infection with oncogenic H-Ras^(V12), leading toOncogene-induced senescence (OIS). At day 9 post DNA damage orintroduction of the oncogene, cells acquired senescent-like morphologyand proliferative arrest (FIG. 1A). At this time point, the cells wereexposed to Sulfo-NHS—SS-biotin, lysed and loaded on columns to capturemembrane proteome. Following a few rounds of washing, the proteins wereeluted to obtain membrane protein samples. The samples were thenanalyzed by mass spectrometry (FIG. 1B). 2198 different proteins wereidentified in the samples, 257 of them existed only in samples derivedfrom senescent cells but not from growing cells (FIG. 1C).

An integrative list of human hits with 5 peptides and more +20% coverageand more is provided in Table 1, herein below.

TABLE 1 Unique Protein Peptides Sequence SEQ Gene Protein Per CoverageAverage ID No. Bank No. Gene Protein Names Description Protein (%) Ratio26 NM_001199954.1. ACTG1 ACTG_HUMAN, Actin, 6 70.7 8.47 SW: P63261cytoplasmic 2 27 NM_001150.2. ANPEP B4DP96_HUMAN, Uncharac- 7 26.3 2.70TR: B4DP96 terized protein 28 NM_001177.5. ARL1 B4DZG7_HUMAN, ADP- 557.0 2.11 TR: B4DZG7 ribosylation factor-like protein 1 29 NM_001693.3.ATP6V1B2 VATB2_HUMAN, V-type proton 8 28.8 4.37 SW: P21281 ATPasesubunit B, brain isoform 30 NM_004859.3. CLTC CLH1_HUMAN, Isoform 2 of43 34.3 2.95 SW: Q00610-2 Clathrin heavy chain 1 31 NM_004094.4. EIF2S1H0YJS4_HUMAN, Eukaryotic 5 23.8 2.52 TR: H0YJS4 translation initiationfactor 2 subunit 1 32 NM_002056.3. GFPT1 GFPT1_HUMAN, Isoform 2 of 819.7 2.29 SW: Q06210-2 Glucosamine- fructose-6- phosphateaminotransferase [isomerizing] 1 33 NM_000424.3. KRT5 K2C5_HUMAN,Keratin, type II 10 20.8 2.98 SW: P13647 cytoskeletal 5 34NM_001256282.1 KRT8 K2C8_HUMAN, Keratin, type II 5 20.5 2.14 SW: P05787cytoskeletal 8 35 XM_005248567.1. MCCC2 MCCB_HUMAN, Isoform 2 of 7 25.02.43 SW: Q9HCC0-2 Methylcrotonoyl-CoA carboxylase beta chain,mitochondrial 36 NM_001114614.1. MFGE8 F5H7N9_HUMAN, Lactadherin 5 29.02.89 TR: F5H7N9 short form 37 NM_001024628.2. NRP1 Q5T7F1_HUMAN,Neuropilin 1 6 27.6 5.49 TR: Q5T7F1 38 NM_201381.2. PLEC PLEC_HUMAN,Isoform 7 106 45.8 1.93 SW: Q15149-7 of Plectin 39 NM_152132.2. PSMA3PSA3_HUMAN, Isoform 2 of 5 25.4 2.18 SW: P25788-2 Proteasome subunitalpha type-3 40 NM_001199163.1. PSMC5 PRS8_HUMAN, Isoform 2 of 8 25.14.72 SW: P62195-2 26S protease regulatory subunit 8 41 NM_012232.5. PTRFPTRF_HUMAN, Isoform 2 of 5 28.3 2.01 SW: Q6NZI2-2 Polymerase I andtranscript release factor 42 NM_004637.5. RAB7A RAB7A_HUMAN, Ras-related5 31.9 2.75 SW: P51149 protein Rab-7a 43 NM_001256577.2. RPL10F8W7C6_HUMAN, 60S ribosomal 6 31.9 3.38 TR: F8W7C6 protein L10 44NM_001042576.1. RRBP1 F8W7S5_HUMAN, Ribosome- 9 24.4 3.70 TR: F8W7S5binding protein 1 45 NM_001788.5 SEPT7 H0Y3Y4_HUMAN, Septin-7 8 21.2214.68 TR: H0Y3Y4 46 NM_006288.3. THY1 E9PIM6_HUMAN, Thy-1 membrane 645.8 3.91 TR: E9PIM6 glycoprotein

In order to understand the nature of the identified proteins,integrative analysis of the identified proteins was performed using thedata mining software, Ingenuity. The distribution of the protein'soriginal subcellular origin was analyzed. Surprisingly, much of thecell-surface proteome was recognized as an intra-cellular (FIG. 1C).Nonetheless, the vast majority of the proteins are presented by only 2-4peptides, which could imply that many peptides identified do not standfor the full and functional proteins, but rather for peptides presentedby antigen presenting machineries. These machineries might include majorhisto-compatibility complexes and members of the heat shock proteinfamilies.

In a parallel analysis approach, the identified proteins were mapped toknown canonical pathways. In the context of immune-related processes,integrin signaling was identified as the strongest hit (FIG. 1D).Integrin signaling in senescent cells consist of at least 10 differentintegrins, predominantly ITGA3 and ITGB5. The second strongestimmunological hit was Fcγ-receptor mediated phagocytosis. This findingis in-line with several studies which identify macrophages and monocytesas potentially important players in immune surveillance of senescentcells [16]. Another strong immunological hit was fMLP (f-Met-Leu-Phe)signaling. N-formylated peptides are believed to derive frommitochondrial proteins upon tissue damage and known to act mainly onneutrophils [17]. fMLP is a strong chemo-attractant, but also inducesadherence, degranulation and production of tissue-destructiveoxygen-derived free radicals in phagocytic cells [17, 18].

Finally, potential upstream regulators were identified (FIG. 1E). Nrf2,the nuclear factor 2 transcription factor, was found to be the mostactivated. Although Nrf2 is constitutively expressed in all tissues, itmay be further induced by cellular stressors including endogenousreactive-oxygen species and oncogene activation [19]. This singletranscription factor mediates multiple avenues of cytoprotection byactivating the transcription of more than 200 genes interacts with otherimportant cell regulators such as p53 and NF-κB and through theircombined interactions is the guardian of health-span, protecting againstmany age-related diseases including cancer and neurodegeneration [20].Conversely, RICTOR, the Rapamycin-insensitive companion of mTOR, wasidentified as being the most inhibited upstream regulator. RICTOR bindsdirectly to mTOR to stabilize TORC2 which regulates cell growth andsurvival in response to hormonal signals and growth factors [21].

Next, the results from the two main experiments were intersected. Thisapproach increased exclusivity of these proteins for the senescenceprogram with no dependence on the exact trigger and increased theconfidence in specific proteins. A short but unique list of 9 proteinswas found to be common between both DIS and OIS (FIG. 2A). Under theserestrictions, 7 out of the 9 proteins were previously documented asbeing found in the plasma membrane compartment, and 3 of them werepreviously reported to exist apically on cell surface (according to theUniProtKB/Swiss-Prot database). A closer look at the top 3 proteinsrevealed that one of them is ICAM1, a cell surface glycoprotein which istypically expressed on endothelial cells and cells of the immune system(FIG. 2B). This protein is known to be up-regulated during cellularsenescence, and therefore it can serve as internal control and provideindependent confirmation for the results of this study [22]. Another hitis ITGA3, an integrin alpha subunit that together with integrin beta-1composes α3β1 integrin duplex. The α3β1 complex is a receptor forvariety of ECM components, known to play a role in neural migration andendothelial adhesion [23, 24]. The third hit, Grp94, also known asEndoplasmin or gp96, is the main ER-resident chaperone. Grp94 belongs tothe heat-shock 90 family and encoded by the HSP90B1 gene in humans. Theencoded protein is localized to the ER where it plays critical roles infolding proteins in the secretory pathway such as Toll-like receptorsand integrins [25]. Unlike the first two hits, normally, this proteindoes not find its way to the cell-surface. Nevertheless, upon severe ERstress, Grp94 can be actively translocated to the extracellularinterface, where it displays important modulatory effects on both theinnate and adaptive immune response [26, 27]. Macrophages for instance,are a prominent GRP94 target, as GRP94 reported to activate severalsignaling pathways in both LPS-dependent and independent manner [28].Second, Grp94 can activate dendritic cells (DCs), to enhance theirimmune-stimulatory capacities [29]. In parallel, Grp94 can undergoendocytosis to antigen-presenting cells (APCs), and to mediate thecross-presentation of Grp94-bound peptides on MHC class I molecules foractivation of cytotoxic T cells [30, 31].

Despite its average size (92469 Da), 42 different unique peptides wereidentified for this protein, covering almost 60% of protein sequence(FIG. 2B). Analysis of the exact location of those sequences on theprotein sequence map suggests that the hits represent the full-lengthprotein and not fragments or single peptides (FIG. 2C).

In order to validate the existence of the fill length Grp94 protein oncell-surface of senescent cells, an unbiased approach was implementedwhich consists of plasma membrane purification followed by Western Blot(WB) analysis. The results of this experiment show that Grp94cell-surface expression is preferential or even exclusive to senescentcells, with a clear band in the size of ˜96 kDA (FIG. 2D). In order toquantitatively assess this elevation, immunofluorescence staining wasperformed on live cells under conditions which allow the staining ofonly the external Grp94 molecules, and the samples were analyzed by FACS(FIG. 2E). The results indicate a 3-fold increase in Grp94 levels onsenescent cells (FIG. 2F). As a positive control the levels of ICAM1were examined which showed a 2-fold increase in senescent cells (FIGS.2G-H). Finally, IMR90 growing or DIS cells were seeded on cover-slipsand stained for cell-surface Grp94/HLA-A,B,C. In agreement with theresults described above, the senescent IMR90 cells display a markedincrease in the levels of cell-surface Grp94 (FIG. 2I).

Stress in the ER might increase its promiscuity, enabling ER residentproteins to be found in other sites in the cell, such as cell-surface.In order to understand whether the existence of Grp94 on the surface ofsenescent cells is a direct consequence of the senescence program, oralternatively, a common result of stress conditions, the presentinventors examined the kinetics of cell-surface Grp94 at differenttime-points after treating propagating IMR90 cells with Etoposide (FIG.3A). Importantly, Grp94 levels were not elevated 3 days after etoposidetreatment, indicating that the presence of Grp94 on cell surface is nota common stress response, but only after the establishment of thesenescent program (FIG. 3B). In addition, the data suggest that duringcellular senescence Grp94 gradually accumulates on the cell surface. Thecontinuous recycling of escaped ER proteins such as Grp94 is mediated byretrograde transport from Golgi to ER through COPI-coated vesicles [32].During this process, Grp94 localization is regulated through itsC-terminal KDEL sequence, which is recognized by the KDEL receptor ERD2[33]. Grp94 homodimerization potentially promote ERD2 binding [34, 35].Consequently, ERD2-Grp94 complex returns to the ER where it dissociates,freeing ERD2 for further cycling of transport. In order to examine thevalidity of this pathway to Grp94 during the senescence program, GPM1, asmall molecule which has been shown to promote dimerization of Grp94,thus promoting recycling of Grp94 into the ER and blocking itstranslocation to cell-surface (FIG. 3B) [36] was synthesized. Afterensuring that GPM1 does not affect the cell's viability under differentconcentrations (FIG. 3C), it was found that GPM1 could reduce cellsurface levels of Grp94 of senescent IMR90 cells (FIG. 3D). Thus, it maybe concluded that GPM1 may be used to manipulate cell-surface levels ofGrp94, and accordingly to investigate the role of Grp94 in theinteraction of senescent cells with the immune system.

Grp94 is known to have multiple effects on the immune system, a propertyfor which it has received the title “the immune system's Swissarmy-knife” [27]. Studies from recent years have revealed closeinteractions between senescent cells and the immune system. Immunesurveillance of senescent cells is mediated on many occasions by NKcells which are attracted to the site of the senescent cells where theyare recognized and eliminated [7, 37]. In order to elucidate possiblerole for Grp94 in immune surveillance of NK cells the total levels ofGr94 in senescent IMR90 cells were down-regulated using specific siRNAfor 4 days. The effect was validated the by Western blot analysis (FIG.4A). A slight decrease in the viability of the siHSP90B1 treated cellswas observed in comparison to siControl (FIG. 4B). This result is inline with the role of ER-resident Grp94 in stabilization of unfoldedproteins during stress conditions. Finally, those cells were co-culturedwith NK-92 cells for 2 hours. The NK cells were then gently removed andcytotoxicity was calculated as the ratio between the IRM90 cell'sviability prior to and following co-culture. The results show adecreased cytotoxicity of NK-92 cells toward senescent cells that havelow Grp94 expression levels (FIG. 4C), suggesting that NK cellcytotoxicity is mediated, at least in part, by cell-surface Grp94.

In order to understand the functional importance of Grp94 forphagocytosis of macrophages, DIS IMR90 cells were treated with GPM1 orDMSO. The viability of senescent cells post co-culturing with human MM6monocytes was examined. The preliminary data show that under theeffective concentration of GPM1 (100 μM) senescent cells have reducedviability following co-culturing with monocytes (FIG. 4D), suggestingGrp94 as a regulator of immune surveillance of senescent cells.

The levels of cell surface Grp94 were also examined in mouse embryonicfibroblast cells (MEFs). A significant elevation in cell surface Grp94was detected upon treatment with etoposide (FIG. 4E). The relativelyhigh correlation between localization of Grp94 in human and mouse cellsimplies that it may serve as a new marker for senescent cells.

In order to shed more light on the biological significance of cellsurface Grp94 in senescent cells, mice were treated with GPM1 followingthe establishment of chronic fibrosis in the liver. It was previouslydemonstrated that senescent hepatic stellate cells are graduallyeliminated by immune cells until their full elimination around 10-20days from the last CCl₄ injection. The administration of GPM1dramatically increased intracellular levels of Grp94 and eliminated thepositive staining at the plasma membrane regions (FIG. 5A). Histologicalanalyses indicate that GPM1 treatment blocks the clearance of senescentcells (FIG. 5B). The increase in tissue-residing senescent cells wasaccompanied with increased fibrosis in the GPM1-treated animals comparedto the Vehicle-treated animals (FIG. 5B). Quantitative analysisconfirmed these findings (FIG. 5C, 5D).

Although the invention has been described in conjunction with specificembodiments thereof, it is evident that many alternatives, modificationsand variations will be apparent to those skilled in the art.Accordingly, it is intended to embrace all such alternatives,modifications and variations that fall within the spirit and broad scopeof the appended claims.

All publications, patents and patent applications mentioned in thisspecification are herein incorporated in their entirety by referenceinto the specification, to the same extent as if each individualpublication, patent or patent application was specifically andindividually indicated to be incorporated herein by reference. Inaddition, citation or identification of any reference in thisapplication shall not be construed as an admission that such referenceis available as prior art to the present invention. To the extent thatsection headings are used, they should not be construed as necessarilylimiting.

REFERENCES

-   1. Campisi, J. and F. d'Adda di Fagagna, Cellular senescence: when    had things happen to good cells. Nat Rev Mol Cell Biol, 2007.    8(9): p. 729-40.-   2. Collado, M., M. A. Blasco, and M. Serrano, Cellular senescence in    cancer aging. Cell, 2007. 130(2): p. 223-33.-   3. Adams, P. D., Healing and hurting: molecular mechanisms,    functions and pathologies of cellular senescence. Mol Cell, 2009.    36(1): p. 2-14.-   4. Serrano, M., et al., Oncogenic ras provokes premature cell    senescence associated with accumulation of p53 and p16INK4a.    Cell, 1997. 88(5): p. 593-602.-   5. Xue, W., et al., Senescence and tumour clearance is triggered by    p53 restoration in murine liver carcinomas. Nature, 2007.    445(7128): p. 656-60.-   6. Kang, T. W., et al., Senescence surveillance of pre-malignant    hepatocytes limits liver cancer development. Nature, 2011.    479(7374): p. 547-51.-   7. Krizhanovsky, V., et al., Senescence o f activated stellate cells    limits liver fibrosis. Cell, 2008. 134(4): p. 657-67.-   8. Michaloglou, C., et al., BRAFE600-associated senescence-like cell    cycle arrest of human naevi. Nature, 2005. 436(7051): p. 720-4.-   9. Parrinello, S., et al., Stromal-epithelial interactions in aging    and cancer: senescent fibroblasts alter epithelial cell    differentiation. J Cell Sci, 2005. 11.8(Pt 3): p. 485-96.-   10. Jeyapalan, J. C. and J. M. Sedivy, Cellular senescence and    organismal aging. Mech Ageing Dev, 2008. 129(7-8): p. 467-74.-   11. Collado, M. and M. Serrano, Senescence in tumours: evidence from    mice and humans. Nat Rev Cancer, 2010. 10(1): p. 51-7.-   12. Ovadya, Y. and V. Krizhanovsky, Senescent cells: SASPected    drivers of age-related pathologies. Biogerontology, 2014. 15(6): p.    627-42.-   13. Grivennikov, S. I., F. R. Greten, and M. Karin, Immunity,    inflammation, and cancer. Cell, 2010. 140(6): p. 883-99.-   14. Baker, D. J., et al., Opposing roles for p16Ink4a and p19Arf in    senescence and ageing caused by BubR1 insufficiency. Nat Cell    Biol, 2008. 10(7): p. 825-36.-   15. Baker, D. J., et al., Clearance of p16Ink4a-positive senescent    cells delays ageing-associated disorders. Nature, 2011.    479(7372): p. 232-6.-   16. Lujambio A., et al., Non-cell-autonomous tumor suppression by    p53. Cell,2013. 153(2): p. 449-60.-   17. Carp, H., Mitochondrial N-formylmethionyl proteins as    chemoattractants for neutrophils. The Journal of experimental    medicine, 1982. 155(1): p. 264-275.-   18. Selvatici, R., et al., Signal transduction pathways triggered by    selective formylpeptide analogues in human neutrophils. Eur J    Pharmacol, 2006. 534(1-3): p. 1-11.-   19. DeNicola, G. M., et al., Oncogene-induced Nrf2 transcription    promotes ROS detoxification and tumorigenesis. Nature, 2011.    475(7354): p. 106-9.-   20. Lewis, K. N., et al., Nrf2, a guardian of healthspan and    gatekeeper of species longevity. Integr Comp Biol, 2010. 50(5): p.    829-43.-   21. Shiota, C., et al., Multiallelic disruption of the rictor gene    in mice reveals that mTOR complex 2 is essential for fetal growth    and viability. Dev Cell, 2006. 11(4): p. 583-9.-   22. Gorgoulis, V. G., et al., p53-dependent ICAM-1 overexpression in    senescent human cells identified in atherosclerotic lesions. Lab    Invest, 2005. 85(4): p. 502-11.-   23. Schmid, R. S., et al., alpha3beta1 integrin modulates neuronal    migration and placement during early stages of cerebral cortical    development. Development, 2004. 131(24): p. 6023-31.-   24. Glinskii, O. V., et al., Endothelial integrin alpha3beta1    stabilizes carbohydrate-mediated tumor/endothelial cell adhesion and    induces macromolecular signaling complex formation at the    endothelial cell membrane. Oncotarget, 2014. 5(5): p. 1382-9.-   25. Wu, S., et al., The molecular chaperone gp96/GRP94 interacts    with Toll-like receptors and integrins via its C-terminal    hydrophobic domain. J Biol Chem, 2012. 287(9): p. 6735-42.-   26. Altmeyer, A., et al., Tumor-specific cell surface expression of    the KDEL containing, endoplasmic reticular heat shock protein gp96.    Int J Cancer, 1996. 69(4): p. 340-9.-   27. Schild, H. and H. G. Rarnmensee, gp96—the immune system's Swiss    army knife. Nat Immunol, 2000. 1(2): p. 100-1.-   28. Reed, R. C., et al., GRP94/gp96 elicits ERK activation in murine    macrophages. A role for endotoxin contamination in NF-kappa B    activation and nitric oxide production. J Biol Chem, 2003.    278(34): p. 31853-60.-   29. Zheng, H., et al., Cell surface targeting of heat shock protein    gp96 induces dendritic cell maturation and antitumor immunity. J    Immunol, 2001. 167(12): p. 6731-5.-   30. Dai, J., et al., Cell surface expression of heat shock protein    gp96 enhances cross-presentation of cellular antigens and the    generation of tumor-specific T cell memory. Cancer Immure, 2003.    3: p. 1.-   31. Berwin, B., et al., Scavenger receptor-A mediates gp96/GRP94 and    calreticulin internalization by antigen-presenting cells. EMBO    J, 2003. 22(22): p. 6127-36.-   32. Yamamoto, K., et al., The KDEL receptor mediates a retrieval    mechanism that contributes to quality control at the endoplasmic    reticulum. EMBO J, 2001. 20(12): p. 3082-91.-   33. Semenza, J. C., et al., ERD2, a yeast gene required for the    receptor-mediated retrieval of luminal ER proteins from the    secretory pathway. Cell, 1990. 61(7): p. 1349-57.-   34. Dollins, D. E., et al., Structures of GRP94-nucleotide complexes    reveal mechanistic differences between the hsp90 chaperones. Mol    Cell, 2007. 28(1): p. 41-56.-   35. Nemoto, T. and N. Sato, Oligomeric forms of the 90-kDa heat    shock protein. Biochem J, 1998. 330 (Pt 2): p. 989-95.-   36. Han, J. M., et al., Identification of gp96 as a novel target for    treatment of autoimmune disease in mice. PLoS One, 2010. 5(3): p.    e9792.-   37. Sagiv, A. and V. Krizhanovsky, Immunosurveillance of senescent    cells: the bright side of the senescence program.    Biogerontology, 2013. 14(6): p. 617-28.-   38. Murray, P. J. and T. A. Wynn, Protective and pathogenic    functions of macrophage subsets. Nat Rev Immunol, 2011. 11(11): p.    723-37.-   39. Binder, R. J. and P. K. Srivastava, Esential role of CD91 in    re-presentation of gp96-chaperoned peptides. Proc Natl Acad Sci    USA, 2004. 101(16): p. 6128-33.-   40. Binder, R. J., D. K. Han, and P. K. Srivastava, CD91: a receptor    fbr heat shock protein gp96. Nat limmunol, 2000. 1(2): p. 151-5.-   41. Naylor, R. M., D. J. Baker, and J. M. van Deursen, Senescent    cells: a novel therapeutic target for aging and age-related    diseases. Clin Pharmacol Ther, 2013. 93(1): p. 105-16.-   42. Mantovani, A., et al., Macrophage plasticity and polarization in    tissue repair and remodelling. J Pathol, 2013. 229(2): p. 176-85.

What is claimed is:
 1. A method of treating a chronic inflammatorydisease associated with cellular senescence in a subject in need, saidmethod comprising administering a pharmaceutical agent to the subject,wherein said pharmaceutical agent is an antibody that binds specificallyto a cell surface polypeptide set forth in SEQ ID NO: 29 or saidpharmaceutical agent is an antibody that binds specifically to a cellsurface polypeptide set forth in SEQ ID NO: 43, wherein said cell is asenescent cell and wherein accumulation of senescent cells is observedin said disease associated with cellular senescence.
 2. The method ofclaim 2, wherein said chronic inflammatory disease associated withcellular senescence is selected from Alzheimer's Disease, diabetes,myocardial infarction, and atherosclerosis; a fibrotic disease includingidiopathic pulmonary fibrosis; a pulmonary disease including chronicobstructive pulmonary disease (COPD); liver fibrosis; a diseaseassociated with bone degeneration; wound healing; an age-relateddisease; or an autoimmune disease.
 3. The method of claim 1, wherein thepresence of said polypeptide on the cell surface is preferentiallypresent on the surface of senescent cells compared with normalnon-senescent cells.